Thiochromane derivatives and their use as thrombin inhibitors

ABSTRACT

There is provided compounds of formulae I and IA                    
     wherein Y, R 1 , R 2 , R 3 , D 1  and D 2  have meanings given in the description which are useful as, or as prodrugs of, competitive inhibitors of trypsin-like proteases, such as thrombin, and in particular in the treatment of conditions where inhibition of thrombin is required (e.g. thrombosis) or as anticoagulants.

This application is a national stage filing under 35 U.S.C. 371 of PCTapplication PCT/SE01/01052, filed May 14, 2001, which claims priorityfrom Sweden Application No. 0001803-6, filed May 16, 2000, thespecifications of each of which are incorporated by reference herein.PCT Application PCT/SE01/01052 was published under PCT Article 21(2) inEnglish.

FIELD OF THE INVENTION

This invention relates to novel pharmaceutically useful compounds, inparticular compounds that are, or are prodrugs of, competitiveinhibitors of trypsin-like serine proteases, especially thrombin, theiruse as medicaments, pharmaceutical compositions containing them andsynthetic routes to their production.

BACKGROUND

Blood coagulation is the key process involved in both haemostasis (i.e.the prevention of blood loss from a damaged vessel) and thrombosis (i.e.the formation of a blood clot in a blood vessel, sometimes leading tovessel obstruction).

Coagulation is the result of a complex series of enzymatic reactions.One of the ultimate steps in this series of reactions is the conversionof the proenzyme prothrombin to the active enzyme thrombin.

Thrombin is known to play a central role in coagulation. It activatesplatelets, leading to platelet aggregation, converts fibrinogen intofibrin monomers, which polymerise spontaneously into fibrin polymers,and activates factor XIII, which in turn crosslinks the polymers to forminsoluble fibrin. Furthermore, thrombin activates factor V and factorVIII leading to a “positive feedback” generation of thrombin fromprothrombin.

By inhibiting the aggregation of platelets and the formation andcrosslinking of fibrin, effective inhibitors of thrombin would beexpected to exhibit antithrombotic activity. In addition, antithromboticactivity would be expected to be enhanced by effective inhibition of thepositive feedback mechanism.

Further, it is known that administration of prodrugs of thrombininhibitors may give rise to improvements in:

(a) certain pharmacokinetic properties after administration of; and

(b) the prevalence of certain side effects associated with, thoseinhibitors.

PRIOR ART

The early development of low molecular weight inhibitors of thrombin hasbeen described by Claesson in Blood Coagul. Fibrinol. (1994) 5, 41 1.

Blombäck et al (in J. Clin. Lab. Invest. 24, suppl. 107, 59, (1969))reported thrombin inhibitors based on the amino acid sequence situatedaround the cleavage site for the fibrinogen chain. Of the amino acidsequences discussed, these authors suggested the tripeptide sequencePhe-Val-Arg (P9-P2-P1, hereinafter referred to as the P3-P2-P1 sequence)would be the most effective inhibitor.

Thrombin inhibitors based on dipeptidyl derivatives with an -aminoalkylguanidine in the P1-position are known from U.S. Pat. No. 4,346,078 andInternational Patent Application WO 93/11152. Similar, structurallyrelated, dipeptidyl derivatives have also been reported. For exampleInternational Patent Application WO 94/29336 discloses compounds with,for example, aminomethyl benzamidines, cyclic aminoalkyl amidines andcyclic aminoalkyl guanidines in the P1-position (International PatentApplication WO 97/23499 discloses prodrugs of certain of thesecompounds); European Patent Application 0 648 780, discloses compoundswith, for example, cyclic aminoalkyl guanidines in the P1-position.

Thrombin inhibitors based on peptidyl derivatives, also having cyclicaminoalkyl guanidines (e.g. either 3- or 4-aminomethyl-1-amidino-piperidine) in the P1-position are known fromEuropean Patent Applications 0 468 231, 0 559 046 and 0 641 779.

Thrombin inhibitors based on tripeptidyl derivatives with argininealdehyde in the P1-position were first disclosed in European PatentApplication 0 185 390.

More recently, arginine aldehyde-based peptidyl derivatives, modified inthe P3-position, have been reported. For example, International PatentApplication WO 93/18060 discloses hydroxy acids, European PatentApplication 0 526 877 des-amino acids, and European Patent Application 0542 525 O-methyl mandelic acids in the P3-position.

Inhibitors of serine proteases (e.g. thrombin) based on electrophilicketones in the P1-position are also known. For example, European PatentApplication 0 195 212 discloses peptidyl -keto esters and amides,European Patent Application 0 362 002 fluoroalkylamide ketones, EuropeanPatent Application 0 364 344 ,,-triketocompounds, and European PatentApplication 0 530 1675 -alkoxy ketone derivatives of arginine in theP1-position.

Other, structurally different, inhibitors of trypsin-like serineproteases based on C-terminal boronic acid derivatives of arginine andisothiouronium analogues thereof are known from European PatentApplication 0 293 881.

More recently, thrombin inhibitors based on peptidyl derivatives havebeen disclosed in European Patent Application 0 669 317 andInternational Patent Applications WO 95/35309, WO 95/23609, WO 96/25426,WO 97/02284, WO 97/46577, WO 96/32110, WO 96/31504, WO 96/03374, WO98/06740, WO 97/49404 and WO 99/29664. Certain prodrugs of thrombininhibitors have been disclosed in WO 97/33576.

WO 98/57932 and WO 00/35869 disclose thrombin inhibitors, and prodrugsof thrombin inhibitors, based on peptidyl derivatives with fused, bi- ortri-cyclic acids at the P3-position.

However, there remains a need for effective inhibitors of trypsin-likeserine proteases, such as thrombin. There is, also a need for compoundsthat have a favourable pharmacokinetic profile (e.g. low clearance), areorally bioavailable, and are selective in inhibiting thrombin over otherserine proteases, in particular those involved in haemostatis. Compoundswhich exhibit competitive inhibitory activity towards thrombin would beexpected to be especially useful as anticoagulants and therefore in thetherapeutic treatment of thrombosis and related disorders.

DISCLOSURE OF THE INVENTION

According to the invention there is provided compounds of formula I,

wherein

Y represents S(O) or S(O)₂;

R¹ represents halo; and

R² represents H, halo or C₁₋₄ alkoxy (which latter group is optionallysubstituted by one or more halo groups);

or a pharmaceutically acceptable derivative thereof, which compounds arereferred to hereinafter as “the compounds of the invention”.

The term “pharmaceutically acceptable derivatives” of compounds offormula I includes pharmaceutically acceptable salts. Suitable saltsinclude inorganic acid (e.g. hydrogen halide), and organic acid (e.g.acetic, methanesulfonic or trifluoroacetic acid), addition salts.

The alkyl part of alkoxy groups which R² may represent, may, when thereis a sufficient number of carbon atoms, be linear or branched, besaturated or unsaturated, be cyclic, acyclic or part cyclic/acyclic,and/or be optionally interrupted by an O atom.

Halo groups which R¹ and R² may represent, and with which R² may besubstituted, include fluoro, chloro, bromo and iodo.

Abbreviations are listed at the end of this specification.

Compounds of the invention that may be mentioned include those in whichY represents S(O)₂.

Preferred compounds of the invention include those in which:

R¹ represents chloro;

R² represents H, halo or C₁₋₂ alkoxy (which latter group is optionallysubstituted by one or more halo (e.g. fluoro) groups).

More preferred compounds of the invention include those in which:

R¹ represents chloro; and

R² represents H, chloro, OCHF₂, OCF₃ or, especially, OCH₃.

Preferred compounds of formula I include the compounds of the Examplesdescribed hereinafter, particularly the compound of Example 1.

According to the invention there is also provided a process for thepreparation of compounds of formula I which comprises:

(i) the coupling of a compound of formula III,

 wherein Y, R¹ and R² are as hereinbefore defined, with4-amidinobenzyl-2-azetidinecarboxamide (see, for example, internationalpatent application WO 97/02284), for example in the presence of acoupling agent (e.g. EDC, DCC, HBTU, HATU, TBTU, PyBOP or oxalylchloride in DMF), an appropriate base (e.g. pyridine, 2,4,6-collidine,DMAP, TEA or DIPEA) and a suitable organic solvent (e.g.dichloromethane, acetonitrile or DMF);

(ii) the coupling of a compound of formula IV,

 wherein Y, R¹ and R² are as hereinbefore defined, withpara-amidino-benzylamine, for example in the presence of a couplingagent (e.g. oxalyl chloride in DMF, EDC, DCC, HBTU, HATU, PyBOP orTBTU), an appropriate base (e.g. pyridine, DMAP, TEA, 2,4,6-collidine orDIPEA) and a suitable organic solvent (e.g. dichloromethane,acetonitrile or DMF); or

(iii) complete oxidation (for compounds of formula I in which Y isS(O)₂), or partial oxidation (for compounds of formula I in which Y isS(O)), of a corresponding compound of formula V,

 wherein R¹ and R² are as hereinbefore defined, for example in thepresence of an appropriate amount of a suitable oxidising agent (e.g.mCPBA, hydrogen peroxide or potassium peroxymonosulfate) and anappropriate organic solvent (e.g. CH₂Cl₂, methanol, water or mixturesthereof), and optionally in the presence of a suitable protic acid (e.g.acetic acid).

The skilled person will appreciate that, in the case of partialoxidation, a mixture of stereoisomers may be obtained, which may beseparated by techniques known to those skilled in the art (e.g. bycolumn chromatography or chiral chromatography).

Compounds of formula III may be prepared by complete or partialoxidation of a compound of formula VI,

wherein R¹ and R² are as hereinbefore defined, for example underconditions such as those described hereinbefore for the synthesis ofcompounds of formula I (process step (iii)).

Compounds of formula IV may be prepared by the coupling of a compound offormula III, as hereinbefore defined, with azetidine-2-carboxylic acid,for example under conditions such as those described hereinbefore forsynthesis of compounds of formula I (see, for example, process steps (i)and (ii)).

Compounds of formula IV may alternatively be prepared by complete orpartial oxidation of a compound of formula VII,

wherein R¹ and R² are as hereinbefore defined, for example underconditions such as those described hereinbefore for the synthesis ofcompounds of formula I (process step (iii)).

Compounds of formula V may be prepared in accordance with peptidecoupling techniques, for example in analogous fashion to the methodsdescribed hereinbefore for compounds of formula I (see, for example,process steps (i) and (ii)). If desired, compounds of formula VII mayalso be prepared in this way.

Compounds of formula VI are available using known and/or standardtechniques.

For example, compounds of formula VI may be prepared by reaction of acompound of formula VIII,

wherein R¹ and R² are as hereinbefore defined, with:

(a) a compound of formula IX,

R″ CN  IX

wherein R″ represents H or (CH₃)₃Si, for example at room, or elevated,temperature (e.g. below 100° C.) in the presence of a suitable organicsolvent (e.g. chloroform or dichloromethane) and, if necessary, in thepresence of a suitable base (e.g. TEA) and/or a suitable catalyst system(e.g. benzylammonium chloride or zinc iodide), followed by hydrolysis inthe presence of an acid (e.g. HCl or H₂SO₄), for example at 20° C. (e.g.according, or analogously, to the method described by C. F. Bigge et al.in J. Med. Chem. (1993) 36, 1977), and then followed by hydrolysis underalkaline conditions (e.g. in the presence of water and either lithium orpotassium hydroxide) to give the free acid;

(b) NaCN or KCN, for example in the presence of NaHSO₃ and water,followed by hydrolysis; or

(c) chloroform, for example at elevated temperature (e.g. above roomtemperature but below 100° C.) in the presence of a suitable base (e.g.sodium hydroxide) and, if necessary, in the presence of a suitablecatalyst system (e.g. benzylammonium chloride), followed by hydrolysis.

The enantiomeric forms of compounds of formula VI (i.e. those compoundshaving different configurations of substituents about the C-atom that isin the α-position relative to the CO₂H group) may be separated bytechniques known to those skilled in the art (e.g. by chromatography,using a chiral chromatographic medium).

Compounds of formula VI may alternatively be prepared by way of aSharpless stereoselective dihydroxylation of a compound of formula X,

wherein R¹ and R² are as hereinbefore defined, under conditions known tothose skilled in the art (e.g. at low temperature (e.g. 0° C.), using,for example, the commercial reagent AD-mix-™ in the presence of suitablesolvent (e.g. t-butanol), followed by oxidation of the resultantintermediate (e.g. at elevated temperature (e.g. 75° C.) in the presenceof a stream of air and Pt/C (5%) in acetone/water).

Compounds of formula VIII are available using known and/or standardtechniques. For example, compounds of formula VIII may be prepared bycyclisation of a compound of formula XI,

wherein L¹ represents a leaving group such as OH, C₁₋₄ alkoxy, C₁₋₄acyloxy (optionally substituted by one or more halo atoms) or halo andR¹ and R² are as hereinbefore defined, for example in the presence of asuitable catalyst (e.g. trifluoroacetic anhydride, a protic acid such asH₂SO₄ or a Lewis acid such as BF₃) and optionally in the presence of anappropriate solvent (e.g. CH₂Cl₂).

Compounds of formula XI may be prepared in accordance with knowntechniques. For example, compounds of formula XI may be prepared byreaction of a compound of formula XII,

wherein R¹ and R² are as hereinbefore defined, with a compound offormula XIII,

wherein L² represents a leaving group such as halo and R^(x) representsC₁₋₆ alkyl, for example at between room and reflux temperature in thepresence of an appropriate base (e.g. triethylamine or Cs₂CO₃) and asuitable solvent (e.g. ethyl acetate or acetone), followed by conversionof the ORX group into an L¹ group under conditions that are well knownto those skilled in the art (e.g. for compounds of formula XI in whichL¹ represents OH, by hydrolysis under alkaline conditions).

Compounds of formulae IX, X, XII and XIII, and derivatives thereof, areeither commercially available, are known in the literature, or may beobtained either by analogy with the processes described herein, or byconventional synthetic procedures, in accordance with standardtechniques, from readily available starting materials using appropriatereagents and reaction conditions (e.g. as described hereinafter).

The compounds of formula I may be isolated from their reaction mixturesusing conventional techniques.

Substituents on the aromatic ring in compounds of formulae I, III, IV,V, VI, VII, VIII, X, XI and XII may be introduced and/or interconvertedusing techniques well known to those skilled in the art. For example,halo may be introduced, both into the aromatic ring and into the alkylpart of alkoxy groups that R² may represent, by reaction with suitablehalogenating agents, for example as described hereinafter.

In accordance with the present invention, pharmaceutically acceptablederivatives of compounds of formula I also include “protected”derivatives of compounds of formula I and/or compounds that act asprodrugs of compounds of formula I.

In this respect, according to a further aspect of the invention there isprovided derivatives of compounds of formula I as defined herein whichare compounds of formula IA,

wherein

Y, R¹ and R² are as hereinbefore defined;

D¹ and D² independently represent H, —OR⁷ or R⁸, or D¹, D² and R³,together with the amidine group to which they are attached, form acyclic group of formula IIa, IIb, IIc, IId or IIe,

 wherein wavy lines indicate the points of attachment to the benzenering;

R³ represents H, or R³, D¹, and D² together with the amidine group towhich they are attached, form a cyclic group of formula IIa, IIb, IIc,IId or IIe;

R⁴ and R⁵ independently represent H or C₁₋₄ alkyl;

R⁶ represents H, C₁₋₆ alkyl (which latter group is optionallysubstituted by one or more halo groups) or C(O)OR¹²;

R⁷ represents H, C₆₋₁₀ aryl, C₁₋₁₀ alkyl (which latter group isoptionally substituted by one or more halo groups), C₁₋₃ alkylphenyl,—C(R^(9a))(R^(9b))R¹⁰, —C(O)R^(11a), —C(O)O¹², —C(O)N(R¹³)R¹⁴ or—(CH₂)_(n)(O)_(m)R¹⁵;

R⁸ represents —C(R^(9a))(R^(9b))R¹⁰, —C(O)R^(11b) or —C(O)OR¹²;

R^(9a) and R^(9b) independently represent, at each occurrence, H or C₁₋₆alkyl;

R¹⁰ represents, at each occurrence, —OC(O)R^(16a), —OC(O)OR¹⁷,—N(R^(18a))C(O)OR¹⁷ or —OC(O)N(R^(18b))R¹⁷;

R^(11a) and R^(11b) independently represent, at each occurrence, C₆₋₁₀aryl, C₁₋₃ alkylphenyl (which latter two groups are optionallysubstituted by one or more substituents selected from C₁₋₆ alkyl andhalo), —[C(R^(19a))(R^(19b))]_(p)OC(O)R^(16b), or R^(11a) representsC₁₋₁₇ alkyl (optionally substituted by C₁₋₆ alkoxy, C₁₋₆ acyloxy, aminoor halo) or R^(11b) represents C₁₋₆ alkyl;

R¹² represents, at each occurrence, C₁₋₁₇ alkyl (optionally substitutedby one or more substituents selected from C₁₋₆ alkoxy, C₁₋₆ acyloxy,—Si(R^(20a))(R^(20b))(R^(21c)) and halo), C₆₋₁₀ aryl, C₁₋₃ alkylphenyl(which latter two groups are optionally substituted by C₁₋₆ alkyl, C₁₋₆alkoxy, cyano or halo), —[C(R^(19a))(R^(19b))]_(q)OC(O)R^(16b) or—CH₂R²¹;

R¹³ represents H or C₁₋₇ alkyl, or together with R¹⁴ represents C₄₋₅alkylene;

R¹⁴ represents C₆₋₁₀ aryl or C₁₋₁₀ alkyl (which latter group isoptionally substituted by one or more substituents selected from OH,halo, CO₂H, C₁₋₆ alkoxy, C₁₋₆ acyloxy and C₆₋₁₀ aryl), or together withR¹³ represents C₄₋₅ alkylene;

R¹⁵ represents C₁₋₇ alkyl optionally substituted by one or more—OC(O)C(H)(R²²)N(G)(G^(a)) groups;

R^(16a), R^(16b) and R¹⁷ independently represent, at each occurrence,C₆₋₁₀ aryl or C₁₋₁₇ alkyl (which latter group is optionally substitutedby one or more substituents selected from —OH, halo, —CO₂H, C₁₋₆ alkoxy,C₁₋₆ acyloxy and C₆₋₁₀ aryl), or R^(16b) represents C₁₋₆ alkoxy(optionally substituted by one or more substituents selected from C₁₋₆alkyl and halo);

R^(11a) and R^(11b) independently represent H or C₁₋₄ alkyl;

R^(19a) and R^(19b) independently represent, at each occurrence, H orC₁₋₆ alkyl;

R^(20a) to R^(20c) independently represent, at each occurrence, C₁₋₆alkyl or phenyl;

R²¹ represents the structural fragment IIf

R²² represents C₃₋₄ alkyl;

G and G^(a) independently represent H, an amino protective group, or Gand

G^(a) together represent an amino protective group;

m represents 0 or 1;

n represents 1, 2 or 3;

p represents 3 or 4;

q represents 2 or 3;

or a pharmaceutically acceptable salt thereof,

provided that:

(a) D¹ and D² do not both represent H; and

(b) when one of D¹ and D² represents —OR⁷, then the other represents H.

Suitable “pharmaceutically acceptable salts” of compounds of formula IAinclude inorganic acid (e.g. hydrogen halide), and organic acid (e.g.acetic, methanesulfonic or trifluoroacetic acid), addition salts.

The compounds of the invention may exhibit tautomerism. All tautomericforms and mixtures thereof are included within the scope of theinvention. Particular tautomeric forms that may be mentioned includethose connected with the position of the double bond in the amidinefunctionality, and the position of the substituent D¹ or D², incompounds of formula IA.

For the avoidance of doubt, in compounds of formula IA, substituents D¹and D² are completely independent of each other. For example, D¹ and D²may both be represented by R⁸ in which, in both cases, R⁸ represents—C(O)R^(11b) in which R^(11b) represents, in both cases, C₁₋₆ alkyl.However, in such instances the C₁₋₆ alkyl group may be the same ofdifferent.

The compounds of formulae I and IA also contain at least two asymmetriccarbon atoms and may therefore exhibit optical and/ordiastereoisomerism. All diastereoisomers may be separated usingconventional techniques, e.g. chromatography or fractionalcrystallisation. The various stereoisomers may be isolated by separationof a racemic or other mixture of the compounds using conventional, e.g.fractional crystallisation or HPLC, techniques. Alternatively thedesired optical isomers may be made by reaction of the appropriateoptically active starting materials under conditions which will notcause racemisation or epimerisation, or by derivatisation, for examplewith a homochiral acid followed by separation of the diastereomericderivatives by conventional means (e.g. HPLC, chromatography oversilica). All stereoisomers are included within the scope of theinvention.

As used herein, the term “aryl” includes phenyl, naphthyl (e.g.2-naphthyl) and the like. Unless otherwise indicated, aryl groups areoptionally substituted by one or more substituents selected form C₁₋₆alkyl and halo.

Alkyl and alkylene groups, as well as alkyl parts of alkoxy, alkylphenyland acyloxy groups, in compounds of formula IA may, when there is asufficient number of carbon atoms, be linear or branched, be saturatedor unsaturated, be cyclic, acyclic or part cyclic/acyclic, and/or beoptionally interrupted by an O atom. The skilled person will appreciatethat, when alkyl groups in compounds of formula IA are cyclic andinterrupted by oxygen, they may then represent oxygen-containingheterocycles such as tetrahydrofuranyl or (where appropriate)tetrahydropyranyl.

Halo groups in compounds of formula IA include fluoro, chloro, bromo andiodo.

As used herein, the term “amino protective group” includes groupsmentioned in “Protective Groups in Organic Synthesis”, 2^(nd) edition, TW Greene & P G M Wutz, Wiley-Interscience (1991), in particular thoseindexed at the start of the chapter entitled “Protection for the AminoGroup” (see pages 309 to 315) of that reference, the disclosure in whichdocument is hereby incorporated by reference.

Specific examples of amino protective groups thus include:

(a) carbamate groups (e.g. methyl, cyclopropylmethyl,1-methyl-1-cyclopropylmethyl, diisopropylmethyl, 9-fluorenylmethyl,9-(2-sulfo)fluorenylmethyl, 2-furanylmethyl, 2,2,2-trichloroethyl,2-haloethyl, 2-trimethylsilylethyl, 2-methylthioethyl,2-methyl-sulfonylethyl, 2(p-toluenesulfonyl)ethyl, 2-phosphonioethyl,1,1-dimethylpropynyl, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)-propyl,1,1-dimethyl-3-(N,N-diethylamino)propyl, 1-methyl-1-(1-adamantyl)ethyl,1-methyl-1-phenylethyl, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl,1-methyl-1-(4-biphenylyl)ethyl, 1-methyl-1-(p-phenylazophenyl)ethyl,1,1-dimethyl-2-haloethyl, 1,1-dimethyl-2,2,2-trichloroethyl,1,1-dimethyl-2-cyanoethyl, isobutyl, t-butyl, t-amyl, cyclobutyl,1-methylcyclobutyl, cyclopentyl, cyclohexyl, 1-methylcyclohexyl,1-adamantyl, isobornyl, vinyl, allyl, cinnamyl, phenyl,2,4,6-tri-t-butylphenyl, m-nitrophenyl, S-phenyl, 8-quinolinyl,N-hydroxypiperidinyl, 4-(1,4-dimethylpiperidinyl),4,5-diphenyl-3-oxazolin-2-one, benzyl, 2,4,6-trimethylbenzyl,p-methoxybenzyl, 3,5-dimethoxybenzyl, p-decyloxybenzyl, p-nitrobenzyl,o-nitrobenzyl, 3,4-dimethoxy-6-nitrobenzyl, p-bromobenzyl, chlorobenzyl,2,4-dichloro-benzyl, p-cyanobenzyl,o-(N,N-dimethylcarboxamidobenzyl)benzyl, m-chloro-p-acyloxybenzyl,p-(dihydroxyboryl)benzyl, p-(phenylazo)benzyl,p-(p′-methoxyphenylazo)benzyl, 5-benzisoxazolylmethyl, 9-anthrylmethyl,diphenylmethyl, phenyl(o-nitrophenyl)methyl, di(2-pyridyl)methyl,1-methyl-1-(4-pyridyl)-ethyl, isonicotinyl or S-benzyl carbamategroups);

(b) amide groups (e.g. N-formyl, N-acetyl, N-chloroacetyl,N-dichloroacetyl, N-trichloroacetyl, N-trifluoroacetyl,N-o-nitrophenylacetyl, N-o-nitrophenoxyacetyl, N-acetoacetyl,N-acetylpyridinium, N-3-phenylpropionyl, N-3-(p-hydroxyphenyl)propionyl,N-3-(o-nitrophenyl)propionyl, N-2-methyl-2-(o-nitrophenoxy)propionyl,N-2-methyl-2-(o-phenylazophenoxy)propionyl, N-4-chlorobutyryl,N-isobutyryl, N-o-nitrocinnamoyl, N-picolinoyl, N-(N′-acetylmethionyl),N-(N′-benzoylphenylalanyl), N-benzoyl, N-p-phenyl-benzoyl,N-p-methoxybenzoyl, N-o-nitrobenzoyl or N-o-(benzoyloxymethyl)benzoylamide groups);

(c) alkyl groups (e.g. N-allyl, N-phenacyl, N-3-acetoxypropyl,N-(4-nitro-1-cyclohexyl-2-oxo-pyrrolin-3-yl), N-methoxymethyl,N-chloroethoxymethyl, N-benzyloxymethyl, N-pivaloyloxymethyl,N-2-tetrahydropyranyl, N-2,4-dinitrophenyl, N-benzyl,N-3,4-dimethoxybenzyl, N-o-nitrobenzyl, N-di(p-methoxyphenyl)methyl,N-triphenylmethyl, N-(p-methoxyphenyl)diphenylmethyl,N-diphenyl-4-pyridylmethyl, N-2-picolyl N′-oxide or N-dibenzosuberylgroups);

(d) phosphinyl and phosphoryl groups (e.g. N-diphenylphosphinyl,N-dimethylthiophosphinyl, N-diphenylthiophosphinyl, N-diethylphosphoryl,N-dibenzylphosphoryl or N-phenylphosphoryl groups);

(e) sulfenyl groups (e.g. N-benzenesulfenyl, N-o-nitrobenzenesulfenyl,N-2,4-dinitrobenzenesulfenyl, N-pentachlorobenzenesulfenyl,N-2-nitro-4-methoxybenzenesulfenyl or N-triphenylmethylsulfenyl groups);

(f) sulfonyl groups (e.g. N-benzenesulfonyl, N-p-methoxybenzenesulfonyl,N-2,4,6-trimethylbenzenesulfonyl, N-toluenesulfonyl, N-benzylsulfonyl,N-p-methylbenzylsulfonyl, N-trifluoromethylsulfonyl orN-phenacylsulfonyl); and

(g) the N-trimethylsilyl group.

Compounds of formulae I and IA in which the fragment

is in the S-configuration are preferred. Compounds of formulae I and IAin which the fragment

is in the R-configuration are preferred.

The wavy lines on the bonds in the above two fragments signify the bondpositions of the fragments.

Compounds of formula IA may be prepared directly from compounds offormula I, in an analogous manner to compounds of formula I, or usingprocedures appropriately adapted from those used to prepare compounds offormula I. In this respect, processes that may be used to preparecompounds of formula IA include:

(1) for compounds of formula IA in which one of D¹ and D² represents—OR⁷ (in which R⁷ represents H, C₆₋₁₀ aryl, C₁₋₁₀ alkyl (which lattergroup is optionally substituted by one or more halo groups) or C₁₋₃alkylphenyl), reaction of a compound of formula XIV,

wherein Y, R¹ and R² are as hereinbefore defined, with a compound offormula XV,

H₂NOR^(a)  XV

wherein R^(a) represents H, C₆₋₁₀ aryl, C₁₋₁₀ alkyl (which latter groupis optionally substituted by one or more halo groups) or C₁₋₃alkylphenyl, for example at between 40 and 60° C., in the presence of asuitable base (e.g. TEA) and an appropriate organic solvent (e.g. THF,CH₃CN, DMF or DMSO), optionally by pre-treating the compound of formulaXIV with gaseous HCl, in the presence of a lower alkyl (e.g. C₁₋₆ alkyl)alcohol (e.g. ethanol) at, for example, 0° C., to form a compound offormula XVI,

wherein R^(b) represents lower (e.g. C₁₋₆) alkyl, such as ethyl, and Y,R¹ and R² are as hereinbefore defined, which compound may be isolated ifdesired;

(2) for compounds of formula IA in which one of D¹ and D² represents—OR⁷ (in which R⁷ represents H, C₆₋₁₀ aryl, C₁₋₁₀ alkyl (which lattergroup is optionally substituted by one or more halo groups) or C₁₋₃alkylphenyl), reaction of a corresponding compound of formula IA, inwhich one of D¹ and D² represents —C(O)OR¹² (e.g.—C(O)O—CH₂CH₂—Si(CH₃)₃) and the other represents H, with a compound offormula XV, as hereinbefore defined, for example at between room andreflux temperature in the presence of an appropriate organic solvent(e.g. THF, CH₃CN, DMF or DMSO), followed by removal of the —C(O)OR¹²group under conditions known to those skilled in the art;

(3) for compounds of formula IA in which D¹ or D² represents R⁸,reaction of a corresponding compound of formula I or a correspondingcompound of formula IA in which D¹ or D² (as appropriate) represents Hwith a compound of formula XVII,

L³—R⁸  XVII

wherein L³ represents a leaving group, such as halo or p-nitrophenoxy,and R⁸ is as hereinbefore defined, for example at between 0° C. and roomtemperature in the presence of a suitable base (e.g. NaOH) and anappropriate organic solvent (e.g. THF) and/or water;

(4) for compounds of formula IA in which one of D¹ and D² represents—OR⁷ (wherein R⁷ does not represent H), reaction of a correspondingcompound of formula IA in which one of D¹ and D² represents —OH with acompound of formula XVIII,

L³—R^(7a)  XVIII

wherein R^(7a) represents R⁷ as hereinbefore defined, except that itdoes not represent H, and L³ is as hereinbefore defined, for example atbetween 0° C. and reflux temperature, optionally in the presence of anappropriate solvent (e.g. DCM, THF, MeCN or DMF) and a suitable base(e.g. Et₃N or pyridine); or

(5) for compounds of formula IA in which one of D¹ and D² represents Hand the other represents —C(R^(9a))(R^(9b))R¹⁰, wherein R¹⁰ represents—OC(O)R^(16a), —OC(O)OR¹⁷ or —OC(O)N(R^(18b))R¹⁷, reaction of acorresponding compound of formula XIX,

wherein Y, R¹, R², R^(9a) and R^(9b) are as hereinbefore defined with acompound of formula XX,

L³—C(O)R^(c)  XX

wherein R^(c) represents R^(16a), —OR¹⁷ or —N(R^(18b))R¹⁷, and L³, R⁶,R¹⁷ and R^(18b) are as hereinbefore defined, for example underconditions described hereinbefore (e.g. as in step (4) above).

Compounds of formula XIV may be prepared in an analogous fashion tocompounds of formula I, for example by the coupling ofp-cyano-benzylamine with a compound of formula IV, as hereinbeforedefined, under conditions such as those described hereinbefore for thesynthesis of compounds of formula I (see, for example, process steps (i)and (ii)).

Compounds of formula XIX may be prepared by reaction of a correspondingcompound of formula I with an excess of a compound of formula XXI,

R^(9a)C(O)R^(9b)  XXI

wherein R^(9a) and R^(9b) are as hereinbefore defined, for example underconditions known to those skilled in the art.

Compounds of formula IA in which D¹, D² and R³, together with theamidine group to which they are attached, represent a group IIa may beprepared by reaction of a corresponding compound of formula IA in whichD¹ represents OH with a compound of formula XXII,

HalC(O)CH(R⁴)Hal  XXII

wherein Hal represents halo (e.g. chloro) and R⁴ is as hereinbeforedefined, for example at room temperature in the presence of a suitablebase (e.g. triethylamine) and an appropriate organic solvent (e.g.dichloromethane), followed by cyclisation of the resultant intermediate,for example by refluxing in the presence of an appropriate base (e.g.sodium hydride) in a suitable organic solvent (e.g. THF).

Compounds of formula IA in which D¹, D² and R³, together with theamidine group to which they are attached, represent a group IIb may beprepared by reaction of a corresponding compound of formula XXIII,

wherein Y, R¹ and R² are as hereinbefore defined with a compound offormula XXIV,

H₂NCH(R⁴)C(O)OR^(b)  XXIV

wherein R^(b) and R⁴ are as hereinbefore defined, for example at roomtemperature in the presence of a suitable base (e.g. triethylamine) andan appropriate organic solvent (e.g. ethanol).

Compounds of formula IA in which D¹, D² and R³, together with theamidine group to which they are attached, represent a group IIc may beprepared by reaction of a corresponding compound of formula IA in whichD¹ represents OH with a compound of formula XXV,

R⁴CHO  XXV

wherein R⁴ is as hereinbefore defined, for example at room temperature,followed by oxidation of the resultant intermediate under conditionsknown to those skilled in the art.

Compounds of formula IA in which D¹, D² and R³, together with theamidine group to which they are attached, represent a group IId may beprepared by cyclisation of a compound of formula XXVI,

wherein Hal is as hereinbefore defined (especially iodo) and Y, R¹, R²R⁴, R⁵ and R⁶ are as hereinbefore defined, for example at roomtemperature in the presence of a suitable base (e.g. DIPEA) and anappropriate solvent (e.g. dichloromethane).

Compounds of formula XXVI may be prepared by reaction of a compound offormula XXVII,

wherein Y, R¹, R² and R⁶ are as hereinbefore defined with a compound offormula XXVIII,

Hal—C(O)OC(R⁴)(R⁵)Hal  XXVIII

wherein Hal, R⁴ and R¹ are as hereinbefore defined for example at roomtemperature in the presence of a suitable base (e.g. triethylamine) andan appropriate organic solvent (e.g. dichloromethane). It is preferredthat, in compounds of formula XXVIII, Hal represents chloro. In suchcases, the intermediate of formula XXVI that is formed comprises achloro group, which, before cyclisation, is preferably converted to aniodo group, using techniques well know to those skilled in the art (e.g.reaction with NaI).

Compounds of formula IA in which D¹, D² and R³, together with theamidine group to which they are attached, represent a group IIe may beprepared by reaction of a corresponding compound of formula XXIX,

wherein Y, R¹, R² and R⁶ are as hereinbefore defined with formaldehydein water, for example at reflux temperature.

Compounds of formulae XV, XVII, XVIII, XX, XXI, XXII, XXIII, XXIV, XXV,XXVII, XXVIII and XXIX and derivatives thereof, are either commerciallyavailable, are known in the literature, or may be obtained either byanalogy with the processes described herein, or by conventionalsynthetic procedures, in accordance with standard techniques, fromreadily available starting materials using appropriate reagents andreaction conditions (e.g. as described hereinafter).

Compounds of formula IA may be isolated from their reaction mixturesusing conventional techniques.

Substituents on the aromatic and/or non-aromatic, carbocyclic andheterocyclic ring(s) in compounds of formulae IA, XIV, XV, XVI, XVII,XVIII, XIX, XX, XXI, XXIII, XXVI, XXVIII and XXIX may be introducedand/or interconverted using techniques well known to those skilled inthe art. For example, hydroxy may be alkylated to give alkoxy oracylated to give acyloxy, and alkoxy and acyloxy may be hydrolysed togive hydroxy.

It will be appreciated by those skilled in the art that in the processesdescribed above (in relation to compounds of formulae I and IA) thefunctional groups of intermediate compounds may need to be protected byprotecting groups.

Functional groups that it is desirable to protect include hydroxy,amino, aldehyde, ketone, 2-hydroxycarboxylic acid and carboxylic acid.Suitable protecting groups for hydroxy include trialkylsilyl ordiarylalkylsilyl groups (e.g. t-butyldimethylsilyl, t-butyldiphenylsilylor trimethylsilyl) and tetrahydropyranyl. Suitable protecting groups forcarboxylic acid include C₁₋₆ alkyl or benzyl esters. Suitable protectinggroups for amino and amidino include t-butyloxycarbonyl,benzyloxycarbonyl or 2-trimethylsilylethoxycarbonyl (Teoc). Amidinonitrogens may also be protected by hydroxy or alkoxy groups, and may beeither mono- or diprotected. Aldehydes and ketones may be protected asacetals and ketals, respectively, by reacting with e.g. ethylene glycol.2-Hydroxy carboxylic acids may be protected by condensing with e.g.acetone.

The protection and deprotection of functional groups may take placebefore or after coupling, or before or after any other reaction in theabovementioned schemes.

Protecting groups may be removed in accordance with techniques that arewell known to those skilled in the art and as described hereinafter.Persons skilled in the art will appreciate that, in order to obtaincompounds of formula I, or formula IA, in an alternative, and, on someoccasions, more convenient, manner, the individual process stepsmentioned hereinbefore may be performed in a different order, and/or theindividual reactions may be performed at a different stage in theoverall route (i.e. substituents may be added to and/or chemicaltransformations performed upon, different intermediates to thosementioned hereinbefore in conjunction with a particular reaction). Thismay negate, or render necessary, the need for protecting groups.

For example, this is particularly true in respect of the synthesis ofcompounds of formula IA. In this case, D¹ or D² groups which do notrepresent H may be introduced at an earlier stage in the overallsynthesis using the process steps described hereinbefore (see, forexample, steps (1) to (5) above). Further, in the synthesis of compoundsof formula I, the —OH group that is in the α-position relative to thecarboxylic acid in compounds of formulae III and IV may need to beprotected prior to the coupling steps described above.

Accordingly, the order and type of chemistry involved will dictate theneed, and type, of protecting groups as well as the sequence foraccomplishing the synthesis.

The use of protecting groups is fully described in “Protective Groups inOrganic Chemistry”, edited by J W F McOmie, Plenum Press (1973), and“Protective Groups in Organic Synthesis”, 3^(rd) edition, T W Greene & PG M Wutz, Wiley-Interscience (1999).

Protected derivatives of compounds of formulae I and IA may be convertedchemically to compounds of formulae I and IA using standard deprotectiontechniques (e.g. hydrogenation).

Medical and Pharmaceutical Use

Compounds of the invention may possess pharmacological activity as such.Compounds of the invention that may possess such activity include, butare not limited to, compounds of formula I.

However, other compounds of the invention (including compounds offormula IA) may not possess such activity, but may be administeredparenterally or orally, and thereafter metabolised in the body to formcompounds that are pharmacologically active (including, but not limitedto, corresponding compounds of formula I). Such compounds (which alsoincludes compounds that may possess some pharmacological activity, butthat activity is appreciably lower than that of the “active” compoundsto which they are metabolised), may therefore be described as “prodrugs”of the active compounds.

Thus, the compounds of the invention are useful because they possesspharmacological activity, and/or are metabolised in the body followingoral or parenteral administration to form compounds which possesspharmacological activity. The compounds of the invention are thereforeindicated as pharmaceuticals.

According to a further aspect of the invention there is thus providedthe compounds of the invention for use as pharmaceuticals.

In particular, compounds of the invention are potent inhibitors ofthrombin either as such and/or (e.g. in the case of prodrugs), aremetabolised following administration to form potent inhibitors ofthrombin, for example as demonstrated in the tests described below.

By “prodrug of a thrombin inhibitor”, we include compounds that form athrombin inhibitor, in an experimentally-detectable amount, and within apredetermined time (e.g. about 1 hour), following oral or parenteraladministration.

The compounds of the invention are thus expected to be useful in thoseconditions where inhibition of thrombin is required.

The compounds of the invention are thus indicated in the treatmentand/or prophylaxis of thrombosis and hypercoagulability in blood andtissues of animals including man.

It is known that hypercoagulability may lead to thrombo-embolicdiseases. Conditions associated with hypercoagulability andthrombo-embolic diseases which may be mentioned include inherited oracquired activated protein C resistance, such as the factor V-mutation(factor V Leiden), and inherited or acquired deficiencies inantithrombin III, protein C, protein S or heparin cofactor II. Otherconditions known to be associated with hypercoagulability andthrombo-embolic disease include circulating antiphospholipid antibodies(Lupus anticoagulant), homocysteinemi, heparin induced thrombocytopeniaand defects in fibrinolysis. The compounds of the invention are thusindicated both in the therapeutic and/or prophylactic treatment of theseconditions.

The compounds of the invention are further indicated in the treatment ofconditions where there is an undesirable excess of thrombin withoutsigns of hypercoagulability, for example in neurodegenerative diseasessuch as Alzheimer's disease.

Particular disease states which may be mentioned include the therapeuticand/or prophylactic treatment of venous thrombosis and pulmonaryembolism, arterial thrombosis (e.g. in myocardial infarction, unstableangina, thrombosis-based stroke and peripheral arterial thrombosis) andsystemic embolism usually from the atrium during arterial fibrillationor from the left ventricle after transmural myocardial infarction orcaused by congestive heart failure.

Moreover, the compounds of the invention are expected to have utility inprophylaxis of re-occlusion (i.e. thrombosis) after thrombolysis,percutaneous trans-luminal angioplasty (PTA) and coronary bypassoperations; the prevention of re-thrombosis after microsurgery andvascular surgery in general.

Further indications include the therapeutic and/or prophylactictreatment of disseminated intravascular coagulation caused by bacteria,multiple trauma, intoxication or any other mechanism; anticoagulanttreatment when blood is in contact with foreign surfaces in the bodysuch as vascular grafts, vascular stents, vascular catheters, mechanicaland biological prosthetic valves or any other medical device; andanticoagulant treatment when blood is in contact with medical devicesoutside the body such as during cardiovascular surgery using aheart-lung machine or in haemodialysis.

In addition to its effects on the coagulation process, thrombin is knownto activate a large number of cells (such as neutrophils, fibroblasts,endothelial cells and smooth muscle cells). Therefore, the compounds ofthe invention may also be useful for the therapeutic and/or prophylactictreatment of idiopathic and adult respiratory distress syndrome,pulmonary fibrosis following treatment with radiation or chemotherapy,septic shock, septicemia, inflammatory responses, which include, but arenot limited to, edema, acute or chronic atherosclerosis such as coronaryarterial disease, cerebral arterial disease, peripheral arterialdisease, reperfusion damage, and restenosis after percutaneoustrans-luminal angioplasty (PTA).

Compounds of the invention that inhibit trypsin and/or thrombin may alsobe useful in the treatment of pancreatitis.

According to a further aspect of the present invention, there isprovided a method of treatment of a condition where inhibition ofthrombin is required which method comprises administration of atherapeutically effective amount of a compound of the invention to aperson suffering from, or susceptible to such a condition.

The compounds of the invention will normally be administered orally,intravenously, subcutaneously, buccally, rectally, dermally, nasally,tracheally, bronchially, by any other parenteral route or viainhalation, in the form of pharmaceutical preparations comprising activecompound either as a free base, or a pharmaceutically acceptablenon-toxic organic or inorganic acid addition salt, in a pharmaceuticallyacceptable dosage form. Depending upon the disorder and patient to betreated and the route of administration, the compositions may beadministered at varying doses.

The compounds of the invention may also be combined and/orco-administered with any antithrombotic agent with a different mechanismof action, such as the antiplatelet agents acetylsalicylic acid,ticlopidine, clopidogrel, thromboxane receptor and/or synthetaseinhibitors, fibrinogen receptor antagonists, prostacyclin mimetics andphosphodiesterase inhibitors, ADP-receptor (P₂T) antagonists andinhibitors of carboxypeptidase U (CPU).

The compounds of the invention may further be combined and/orco-administered with thrombolytics such as tissue plasminogen activator(natural, recombinant or modified), streptokinase, urokinase,prourokinase, anisoylated plasminogen-streptokinase activator complex(APSAC), animal salivary gland plasminogen activators, and the like, inthe treatment of thrombotic diseases, in particular myocardialinfarction.

According to a further aspect of the invention there is thus provided apharmaceutical formulation including a compound of the invention, inadmixture with a pharmaceutically acceptable adjuvant, diluent orcarrier.

Suitable daily doses of the compounds of the invention in therapeutictreatment of humans are about 0.001-100 mg/kg body weight at peroraladministration and 0.001-50 mg/kg body weight at parenteraladministration.

The compounds of the invention have the advantage that they may, or maybe metabolised to compounds that may, be more efficacious, be lesstoxic, be longer acting, have a broader range of activity, be morepotent, produce fewer side effects, be more easily absorbed, or have abetter pharmacokinetic profile, than, or have other usefulpharmacological, physical, or chemical, properties over, compounds knownin the prior art.

Biological Tests

The following test procedures may be employed.

Test A

Determination of Thrombin Clotting Time (TT)

The inhibitor solution (25 L) is incubated with plasma (25 L) for threeminutes. Human thrombin (T 6769; Sigma Chem. Co or HematologicTechnologies) in buffer solution, pH 7.4 (25 L, 4.0 NIH units/mL), isthen added and the clotting time measured in an automatic device (KC 10;Amelung).

The thrombin clotting time (TT) is expressed as absolute values(seconds) as well as the ratio of TT without inhibitor (TT₀) to TT withinhibitor (TT_(i)). The latter ratios (range 1-0) are plotted againstthe concentration of inhibitor (log transformed) and fitted to sigmoidaldose-response curves according to the equation

y=a/[1+(x/IC ₅₀)^(s)]

where: a=maximum range, i.e. 1; s=slope of the dose-response curve; andIC₅₀=the concentration of inhibitor that doubles the clotting time. Thecalculations are processed on a PC using the software program GraFitVersion 3, setting equation equal to: Start at 0, define end=1(Erithacus Software, Robin Leatherbarrow, Imperial College of Science,London, UK).

Test B

Determination of Thrombin Inhibition With a Chromogenic, Robotic Assay

The thrombin inhibitor potency is measured with a chromogenic substratemethod, in a Plato 3300 robotic microplate processor (Rosys AG, CH-8634Hombrechtikon, Switzerland), using 96-well, half volume microtitreplates (Costar, Cambridge, Mass., USA; Cat No 3690). Stock solutions oftest substance in DMSO (72 L), 0.1−1 mmol/L, are diluted serially 1:3(24+48 L) with DMSO to obtain ten different concentrations, which areanalysed as samples in the assay. 2 L of test sample is diluted with 124L assay buffer, 12 L of chromogenic substrate solution (S-2366,Chromogenix, Mölndal, Sweden) in assay buffer and finally 12 L of-thrombin solution (Human (α-thrombin, Sigma Chemical Co. or HematologicTechnologies) in assay buffer, are added, and the samples mixed. Thefinal assay concentrations are: test substance 0.00068−13.3 mol/L,S-2366 0.30 mmol/L, -thrombin 0.020 NIHU/mL. The linear absorbanceincrement during 40 minutes incubation at 37° C. is used for calculationof percentage inhibition for the test samples, as compared to blankswithout inhibitor. The IC₅₀-robotic value, corresponding to theinhibitor concentration which causes 50% inhibition of the thrombinactivity, is calculated from a log concentration vs. % inhibition curve.

Test C

Determination of the Inhibition Constant K_(i) for Human Thrombin

K_(i)-determinations are made using a chromogenic substrate method,performed at 37° C. on a Cobas Bio centrifugal analyser (Roche, Basel,Switzerland). Residual enzyme activity after incubation of human-thrombin with various concentrations of test compound is determined atthree different substrate concentrations, and is measured as the changein optical absorbance at 405 nm.

Test compound solutions (100 L; normally in buffer or saline containingBSA 10 g/L) are mixed with 200 L of human -thrombin (Sigma Chemical Co)in assay buffer (0.05 mol/L Tris-HCl pH 7.4, ionic strength 0.15adjusted with NaCl) containing BSA (10 g/L), and analysed as samples inthe Cobas Bio. A 60 L sample, together with 20 L of water, is added to320 L of the substrate S-2238 (Chromogenix AB, Mölndal, Sweden) in assaybuffer, and the absorbance change (A/min) is monitored. The finalconcentrations of S-2238 are 16, 24 and 50 μmol/L and of thrombin 0.125NIH U/mL.

The steady state reaction rate is used to construct Dixon plots, i.e.diagrams of inhibitor concentration vs. 1/(A/min). For reversible,competitive inhibitors, the data points for the different substrateconcentrations typically form straight lines which intercept atx=-K_(i).

Test D

Determination of Activated Partial Thromboplastin Time (APTT)

APTT is determined in pooled normal human citrated plasma with thereagent PTT Automated 5 manufactured by Stago. The inhibitors are addedto the plasma (10 L inhibitor solution to 90 L plasma) and incubatedwith the APTT reagent for 3 minutes followed by the addition of 100 L ofcalcium chloride solution (0.025 M) and APTT is determined by use of thecoagulation analyser KC10 (Amelung) according to the instructions of thereagent producer.

The clotting time is expressed as absolute values (seconds) as well asthe ratio of APTT without inhibitor (APTT₀) to APTT with inhibitor(APTT₁). The latter ratios (range 1-0) are plotted against theconcentration of inhibitor (log transformed) and fitted to sigmoidaldose-response curves according to the equation

y=a/[1+(x/IC ₅₀)^(s)]

where: a=maximum range, i.e. 1; s=slope of the dose-response curve; andIC₅₀=the concentration of inhibitor that doubles the clotting time. Thecalculations are processed on a PC using the software program GraFitVersion 3, setting equation equal to: Start at 0, define end=1(Erithacus Software, Robin Leatherbarrow, Imperial College of Science,London, UK). IC₅₀APTT is defined as the concentration of inhibitor inhuman plasma that doubled the Activated Partial Thromboplastin Time.

Test E

Determination of Thrombin Time ex vivo

The inhibition of thrombin after oral or parenteral administration ofthe compounds of formula I, dissolved in ethanol:Solutol3:water(5:5:90), is examined in conscious rats which, one or two days prior tothe experiment, are equipped with a catheter for blood sampling from thecarotid artery. On the experimental day blood samples are withdrawn atfixed times after the administration of the compound into plastic tubescontaining 1 part sodium citrate solution (0.13 mol per L) and 9 partsof blood. The tubes are centrifuged to obtain platelet poor plasma. Theplasma is used for determination of thrombin time or ecarin clottingtime (ECT) as described below.

The citrated rat plasma, 100 L, is diluted with a saline solution, 0.9%,100 L, and plasma coagulation is started by the addition of humanthrombin (T 6769, Sigma Chem Co, USA or Hematologic Technologies) in abuffer solution, pH 7.4, 100 L, or ecarin (Pentapharm). The clottingtime is measured in an automatic device (KC 10, Amelung, Germany).

Where a “prodrug” compound of formula I (e.g. of formula IA) isadministered, concentrations of the appropriate active thrombininhibitor of formula I in the rat plasma are estimated by the use ofstandard curves relating the thrombin time or ecarin clotting time inthe pooled citrated rat plasma to known concentrations of thecorresponding “active” thrombin inhibitor dissolved in saline.

Based on the estimated plasma concentrations of the active thrombininhibitor (which assumes that thrombin time or ECT prolongation iscaused by the aforementioned compound) in the rat, the area under thecurve after oral and/or parenteral administration of the correspondingprodrug compound of formula I is calculated (AUCpd) using thetrapezoidal rule and extrapolation of data to infinity.

The bioavailability of the active thrombin inhibitor after oral orparenteral administration of the prodrug is calculated as below:

[(AUCpd/dose)/(AUCactive,parenteral/dose]×100

where AUCactive,parenteral represents the AUC obtained after parenteraladministration of the corresponding active thrombin inhibitor toconscious rats as described above.

Test F

Determination of Thrombin Time in Urine ex vivo

The amount of the “active” thrombin inhibitor that is excreted in urineafter oral or parenteral administration of “prodrug” compounds of theinvention, dissolved in ethanol:Solutol3:water (5:5:90), is estimated bydetermination of the thrombin time in urine ex vivo (assuming thatthrombin time prolongation is caused by the aforementioned compound).Conscious rats are placed in metabolism cages, allowing separatecollection of urine and faeces, for 24 hours following oraladministration of compounds of the invention. The thrombin time isdetermined on the collected urine as described below.

Pooled normal citrated human plasma (100 L) is incubated with theconcentrated rat urine, or saline dilutions thereof, for one minute.Plasma coagulation is then initiated by the administration of humanthrombin (T 6769, Sigma Chem Company) in buffer solution (pH 7.4; 100L). The clotting time is measured in an automatic device (KC 10;Amelung).

The concentrations of the active thrombin inhibitor in the rat urine areestimated by the use of standard curves relating the thrombin time inthe pooled normal citrated human plasma to known concentrations of theaforementioned active thrombin inhibitor dissolved in concentrated raturine (or saline dilutions thereof). By multiplying the total rat urineproduction over the 24 hour period with the estimated mean concentrationof the aforementioned active inhibitor in the urine, the amount of theactive inhibitor excreted in the urine (AMOUNTpd) can be calculated.

The bioavailability of the active thrombin inhibitor after oral orparenteral administration of the prodrug is calculated as below:

[(AMOUNTpd/dose)/(AMOUNTactive,parenteral/dose]×100

where AMOUNTactive,parenteral represents the amount excreted in theurine after parenteral administration of the corresponding activethrombin inhibitor to conscious rats as described above.

Test G

Metabolic Activation of Prodrug Compounds in vitro

Prodrug compounds of formula IA are incubated at 37° C. with livermicrosomes or 10 000 g (referring to the centrifuge speed) supernatantfractions (i.e. s9 fraction) prepared from human or rat liverhomogenate. The total protein concentration in the incubations are 1 or3 mg/mL dissolved in 0.05 mol/L TRIS buffer (pH 7.4), and with thecofactors NADH (2.5 mmol/L) and NADPH (0. 8 mmol/L) present. The totalvolume of the incubate is 1.2 mL. The initial prodrug concentrations are5 or 10 mol/L. Samples are collected from the incubate at regular isintervals more than 60 minutes after the start of the incubations.Samples (25 L) from the incubate are mixed with an equal volume of humanor rat plasma and an appropriate amount of thrombin, and the clottingtime (i.e. thrombin time) is measured on a coagulometer (KC 10;Amelung). The amount of “active” thrombin inhibitor formed is estimatedby the use of standard curves relating the thrombin time in pooledcitrated human or rat plasma to known concentrations of thecorresponding “active thrombin inhibitor”.

The amount of “active” thrombin inhibitor is alternatively, or inaddition to the above-mentioned method, estimated by the use of LC-MS.

Test H

Determination of Plasma Clearance in Rat

Plasma clearance was estimated in male Sprague Dawley rats. The compoundwas dissolved in water and administered as a subcutaneous bolusinjection at a dose of 4 μmol/kg. Blood samples were collected atfrequent intervals up to 5 hours after drug administration. Bloodsamples were centrifuged and plasma was separated from the blood cellsand transferred to vials containing citrate (10% final concentration).The ecarin clotting time (ECT) was then determined in each plasma sampleby the use of a coagulometer (KC10; Amelung). The plasma concentrationin each sample was determined by the use of a standard curve relatingthe ECT in pooled citrated plasma samples to known concentrations of thecompound. The area under the plasma concentration-time profile wasestimated using the log/linear trapezoidal rule and extrapolated toinfinite time. Plasma clearance (CL) of the compound was then determinedas

CL=Dose/AUC

The values are reported in mL/min/kg.

Test I

Determination of in vitro Stability of Active Thrombin Inhibitors

Liver microsomes were prepared from Sprague-Dawley rats and human liversamples according to internal SOPs. The compounds were incubated at 37°C. at a total microsome protein concentration of 3 mg/mL in a 0.05 mol/LTRIS buffer at pH 7.4, in the presence of the cofactors NADH (2.5mmol/L) and NADPH (0.8 mmol/L). The initial concentration of compoundwas 5 or 10 μmol/L. Samples were taken for analysis up to 60 minutesafter the start of the incubation. The enzymatic activity in thecollected sample was immediately stopped by adding 20% myristic acid ata volume corresponding to 3.3% of the total sample volume. Theconcentration of compound remaining (FINAL CONC) in the 60 min. samplewas determined by means of LCMS using a sample collected at zero time asreference (START CONC). The % of degraded thrombin inhibitor wascalculated as:$100\% \times \frac{\text{[START~~CONC]} - \text{[FINAL~~CONC]}}{\text{[START~~CONC]}}$

Test J

Arterial Thrombosis Model

Vessel damage was induced by applying ferric chloride (FeCl₃) topicallyto the carotid artery. Rats are anaesthetised with an intraperitonealinjection of sodium pentobarbital (80 mg/kg; Apoteksbolaget; Umea,Sweden), followed by continuous infusion (12 mg/kg/h) throughout theexperiment. Rat body temperature was maintained at 38° C. throughout theexperiment by external heating. The experiment started with a 5 minutescontrol period. Five minutes later, human ¹²⁵I-fibrinogen (80 kBq; IM53;Amersham International, Buckinghamshire, UK) was given intravenously andwas used as a marker for the subsequent incorporation of fibrin(ogen)into the thrombus. The proximal end of the carotid artery segment wasplaced in a plastic tube (6 mm; Silastic®; Dow Corning, Mich., USA)opened lengthways, containing FeCl₃-soaked (2 μl; 55% w/w; Merck,Darmstadt, Germany) filter paper (diameter 3 mm; IF; Munktell, Grycksbo,Sweden). The left carotid artery was exposed to FeCl₃ for 10 minutes andwas then removed from the plastic tube and soaked in saline. Fiftyminutes later, the carotid artery was removed and rinsed in saline.Reference blood samples were also taken for determination of blood¹²⁵I-activity, 10 minutes after the injection of 125I -fibrinogen, andat the end of the experiment. The ¹²⁵I-activity in the reference bloodsamples and the vessel segment were measured in a gamma counter (1282Compugamma; LKB Wallac Oy, Turku, Finland) on the same day as theexperiment was performed. The thrombus size was determined as the amountof ¹²⁵I-activity incorporated in the vessel segment in relation to the¹²⁵I-activity in the blood (cpm/mg).

EXAMPLES

The invention is illustrated by way of the following examples. The aminoacid Aze is defined as the S-isomer if not otherwise specified. Theexamples were obtained as diastereoisomers if not otherwise specified.

Example 1(R)-6-Chloro-4-hydroxy-8-methoxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-Pab

(i) 3-(4-Chloro-2-methoxyphenylthio)propanoic Acid, Ethyl Ester

4-Chloro-2-methoxythiophenol (5.78 g, 33 mmol), triethylamine (5.0 mL,36 mmol) and ethyl 3-bromopropanoate (4.6 mL, 36 mmol) were dissolved inethyl acetate (50 mL) and refluxed for 2 h. Water was added and theliquid phases were separated. The aqueous phase was extracted two timeswith ethyl acetate. The combined organic fractions were washed with 1 Maq. HCl and then concentrated in vacuo. Purification by flashchromatography (SiO₂; heptane: dichloromethane (1:1, 0:1)) afforded thesub-title compound as a yellow solid. Yield: 3.0 g (30%). Estimatedpurity: 75% (NMR).

Bis(4-chloro-2-methoxyphenyl) disulfide (1.7 g, 4.9 mmol) was alsoisolated (vide supra). This material was dissolved in THF:EtOH (80 mL of1:1) and stirred under N₂(g). A solution of sodium borohydride (0.38 g,10 mmol) in 2 M aq. NaOH (2 mL) and water (10 mL) was added. The yellowcolour disappeared. After 1 h at room temperature ethyl3-bromopropanoate (1.3 mL, 10 mmol) was added, resulting in evolution ofgas and a white precipitate. After stirring overnight, the solvent wasremoved in vacuo and the residue transferred to a separatory funnel withwater and dichloromethane. The organic phase was dried (MgSO₄) andconcentrated in vacuo affording an additional 2.36 g (88%) of thesub-title compound, a yellow oil that solidified after a few hours. ¹HNMR (400 MHz; CDCl₃): δ 7.29 (d, 1H), 6.94 (dd, 1H), 6.87 (d, 1H), 4.17(q, 2H), 3.92 (s, 3H), 3.15 (t, 2H), 2.61 (t, 2H), 1.18 (t, 3H).

(ii) 3-(4-Chloro-2-methoxyphenylthio)propanoic Acid

To an ice/water-cooled solution of3-(4-chloro-2-methoxyphenylthio)-propanoic acid, ethyl ester (2.31 g,8.41 mmol; see step (i) above) in THF (50 mL) was added a solution oflithium hydroxide monohydrate (0.43 g, 10 mmol) in water (25 mL). Thetemperature was allowed to slowly rise to ambient overnight. THF wasremoved in vacuo, the residue was washed with diethyl ether, acidifiedwith 2 M aq. HCl and then extracted with diethyl ether. The combinedether fractions were extracted with sat. aq. sodium hydrogencarbonate.Careful acidification with aq. HCl afforded a precipitate that wasfiltered off and air-dried.

Yield: 1.71 g (82%). MS(m/z) 245 (M−1)⁻.

(iii) 6-Chloro-8-methoxy-4-thiochromanone

To a solution/suspension of 3-(4-chloro-2-methoxyphenylthio)propanoicacid (1.46 g, 5.92 mmol; see step (ii) above) in dichloromethane (50 mL)under N₂(g) was added boron trifluoride dimethyl etherate (1.4 mL, 15mmol) and trifluoroacetic anhydride (2.1 mL, 15 mmol). After 2.5 h at rtthe reaction was quenched with water (2 mL), then washed with morewater. The deep green colour disappeared when it was further washed withsat. aq. sodium hydrogencarbonate. Drying (MgSO₄) and removal of thesolvent in vacuo afforded the sub-title compound as a yellow solid.

Yield: 1.19g(88%) ¹H NMR (300 MHz; CDCl₃) δ 7.73 (d, 1H), 6.92 (d, 1H),3.91 (s, 3H), 3.21 (m, 2H), 2.95 (m, 2H).

(iv) 6-Chloro-4-hydroxy-8-methoxythiochromane-4-yl-carboxylic Acid,Ethyl Ester

To a stirred solution/suspension of 6-chloro-8-methoxy-4-thiochromanone(2.24 g; 9.79 mmol; see step (iii) above) and zinc iodide (0.078 g; 0.24mmol) in dry methylene chloride (20 mL) under N₂(g) was addedtrimethylsilyl cyanide (1.35 mL; 10.1 mmol). After 2 days at roomtemperature, the reaction mixture was slowly added to stirredice/water-cooled absolute ethanol (50 mL), presaturated with HCl(g) at0° C. After this addition, the cooling bath was removed and the reactionwas monitored using reversed-phase HPLC. After 2 h at rt, the solventswere removed in vacuo. The residue was dissolved in THF (40 mL) andH₂SO₄ (aq.; 0.5 M; 40 mL) and stirred overnight. After concentrationunder reduced pressure (THF removed), extraction with EtOAc and removalof the solvents in vacuo, 3.7 g of crude oil was obtained. Purificationusing preparative reversed-phase HPLC (acetonitrile:0.1 M aq. ammoniumacetate) afforded, after extraction of the appropriate fractions withmethylene chloride, the sub-title compound as an oil that slowlycrystallized.

Yield: 2.26 g (76%) ¹H NMR (300 MHz; CDCl₃) δ 6.80 (d, 1H), 6.73 (d,1H), 4.20-4.35 (m, 2H), 3.98 (s, 1H), 3.87 (s, 3H), 3.19 (m, 1H), 2.98(m, 1H), 2.27-2.42 (m, 2H), 1.23 (t, 3H).

(v) 6-Chloro-4-hydroxy-8-methoxythiochromane-4-yl-carboxylic Acid

To an ice/water-cooled solution of6-chloro-4-hydroxy-8-methoxythiochromane-4-yl-carboxylic acid, ethylester (2.26 g, 7.46 mmol; see step (iv) above) in THF (20 mL) was addeda solution of lithium hydroxide monohydrate (0.68 g, 16 mmol) in water(10 mL). The temperature was allowed to slowly rise to ambientovernight. THF was removed in vacuo, the residue was washed with diethylether, acidified with 2 M aq. HCl and then extracted with ethyl acetate.Drying (MgSO₄) and removal of the solvent in vacuo afforded thesub-title compound.

Yield: 1.53 g (75%). MS(m/z) 273 (M−1)⁻.

(vi) (R)-6-Chloro-4-hydroxy-8-methoxythiochromane-4-yl-carboxylic Acid

The enantiomers of6-chloro-4-hydroxy-8-methoxythiochromane-4-yl-carboxylic acid (3.00 g,10.9 mmol; see step (v) above) were separated using chiralchromatography (Kromasil TBB; heptane:ethyl acetate:formnic acid)affording 1.13 g of the faster moving, not wanted S-enantiomer, and 1.19g (79%) of the slower moving sub-title compound. ee=94.6%

¹H NMR (500 MHz; CD₃OD) δ 6.97 (d, 1H), 6.83 (d, 1H), 3.83 (s, 3H), 3.07(m, 1H), 2.98 (m, 1H), 2.40 (m, 1H), 2.27 (m, 1H).

(vii)(R)-6-Chloro-4-hydroxy-8-methoxy-1,1-dioxothiochromane-4-yl-carboxylicAcid

A solution of(R)-6-chloro-4-hydroxy-8-methoxythiochromane-4-yl-carboxylic acid (0.55g, 2.0 mmol; from step (vi) above) and aqueous hydrogen peroxide (1.0 mLof 35%, 10 mmol) in acetic acid (20 mL) was stirred overnight at ambienttemperature. The solvent was removed in vacuo, after which the residuewas dissolved in water and then freeze dried. Yield: 0.63 g (100%).

MS(m/z) 305 (M−1)⁻. ¹H NMR (400 MHz; CD₃OD) δ 7.23 (d, 1H), 7.06 (d,1H), 3.96 (s, 3H), 3.61 (m, 2H), 2.85 (m, 1H), 2.53 (m, 1H).

(viii)(R)-6-Chloro-4-hydroxy-8-methoxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-Pab(Teoc)

HATU (0.94 g; 2.5 mmol) was added to a solution of(R)-6-chloro-4-hydroxy-8-methoxy-1,1-dioxothiochromane-4-yl-carboxylicacid (0.63 g; 2.05 mmol; see step (vii) above) in DMF (10 mL) at 0° C.After 1 h, a solution of H-Aze-Pab(Teoc)x2 HCl (1.1 g; 2.5 mmol; seeinternational patent application WO 98/57932) andN,N-diisopropylethylamine (1.4 mL; 8.0 mmol) in DMF (5 mL) was addeddropwise. The temperature was allowed slowly to rise to ambientovernight. The solvents were removed in vacuo and the residue waspurified using reversed-phase HPLC (acetonitrile:0.1 M aq. ammoniumacetate). Freeze drying of the appropriate fractions afforded thesub-title compound as a colourless solid.

Yield: 0.50 g (37%) MS(m/z) 663 (M−1)⁻; 665 (M +1)⁺.

(An alternative method of obtaining(R)-6-chloro-4-hydroxy-8-methoxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-Pab(Teoc)is as follows: HATU (0.47 g, 1.2 mmol) was added to a solution of(R)-6-chloro-4-hydroxy-8-methoxythiochromane-4-yl-carboxylic acid (0.31g, 1.1 mmol; see step (vi) above) in DMF (7 mL) at 0° C. After 1 h, asolution of H-Aze-Pab(Teoc)x2HCl (0.61 g, 1.4 mmol; prepared asdescribed in international patent application WO 98/57932) and2,4,6-collidine (0.67 mL, 5.1 mmol) in DMF (9 mL) was added dropwise.After 2 h the reaction mixture was put in a freezer overnight. Thesolvent was removed in vacuo, the residue dissolved in ethyl acetate andthen washed with aq. sat. sodium hydrogencarbonate. Purification usingreversed-phase HPLC (acetonitrile: 0.1 M aq. ammonium acetate) afforded,after freeze-drying of the appropriate fractions, the sub-title compoundas a colourless solid. To an ice/water-cooled solution of the(R)-6-chloro-4-hydroxy-8-methoxythiochromane-4-yl-C(O)-Aze-Pab(Teoc) soobtained (0.25 g, 0.39 mmol), in dichloromethane (20 mL), was addedm-chloroperbenzoic acid (0.25 g of ˜60%, 0.86 mmol), dissolved in asmall volume of dichloromethane. The reaction mixture stirred for 2.5 h,then washed with sat. aq. sodium hydrogencarbonate, concentrated invacuo and purified using reversed-phase HPLC (acetonitrile: 0.1 M aq.ammonium acetate). The fractions of interest were concentrated andextracted with ethyl acetate. Drying (MgSO₄) and removal of the solventin vacuo afforded the sub-title compound. Yield: 0.201 g (76%).)

(ix)(R)-6-Chloro-4-hydroxy-8-methoxy-1,1-dioxothiochromane-4-yl-C(O)Aze-PabxHOAc

To a solution of(R)-6-chloro-4-hydroxy-8-methoxy-1,1-dioxothiochromane-4-yl—C(O)-Aze-Pab(Teoc)(0.092 g, 0.14 mmol; see step (viii) above) in THF (15 mL) was addedtetrabutylammonium fluoride (0.18 mL of 1.0 M, 0.18 mmol). The solutionwas stirred at 50° C. for 20 h, at 60° C. for 5 h, then left at rtovernight. The solvent was removed in vacuo. The residue was purifiedusing reversed-phase HPLC (acetonitrile: 0.1 M aq. ammonium acetate)affording the title compound as a white solid, after freeze-drying theappropriate fractions.

Yield: 0.044 g (50%). MS(m/z) 519 (M−1)³¹ , 521 (M+1)⁺.

¹H NMR (600 MHz; CD₃OD): (complex due to diastereomers/rotamers) δ 7.76(d, 0.9H, rotamer); 7.66 (d, 1.1H, rotamer); 7.54 (d, 0.9H, rotamer);7.47 (d, 1.1H, rotamer); 7.12-7.25 (several peaks, 2H); 5.54 (dd, 0.45H,rotamer); 4.54-4.65 (several peaks, 1.55H); 4.43-4.51 (several peaks,1H); 4.30 (m, 0.55H, rotamer); 4.09 (m, 0.45H, rotamer); 3.99 (m, 0.45H,rotamer); 3.92 (m, 0.55H, rotamer); 3.90 (s, 3H); 3.76 (m, 0.45H,rotamer); 3.60 (m, 1.1H, rotamer); 3.40 (m, 0.45H, rotamer); 2.70-2.88(several peaks, 1.45H); 2.46-2.61 (several peaks, 1.55H); 2.28 (m,0.55H, rotamer); 2.14 (m, 0.45H, rotamer); 1.90 (s, 3H).

¹³C NMR (75 MHz; CD₃OD): (complex due to diastereomers/rotamers,carbonyl and/or amidine carbons) δ 179.8; 174.2; 173.8; 172.9; 168.1.

Example 2(R)-6,8-Dichloro-4-hydroxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-Pab

(i) 3-(2,4-Dichlorophenylthio)propanoic Acid, Ethyl Ester

2,4-Dichlorothiophenol (5.04 g, 28.2 mmol), caesium carbonate (10.1 g,31 mmol) and ethyl 3-bromopropanoate (4.2 mL, 33 mmol) in acetone (100mL) were refluxed overnight under N₂(g). After filtration andconcentration under reduced pressure, the residue was partitionedbetween dichloromethane and water. After separation, the aqueous phasewas extracted once with dichloromethane. Drying and removal of thesolvent in vacuo afforded a liquid residue that contained ˜30% (HPLC) ofunreacted 2,4-dichlorothiophenol. The mixture was refluxed again withcaesium carbonate (4.7 g) and ethyl 3-bromopropanoate (2 mL) inacetonitrile (100 mL) under N₂(g) for 2 h. After filtration andconcentration under reduced pressure, the residue was dissolved indiethyl ether and washed with 2 M aq. sodium hydroxide and water. Dryingand removal of the solvent in vacuo afforded the sub-title compound.

15 Yield: 5.85 g (74%). ¹H NMR (300 MHz; CDCl₃): δ 7.39 (d, 1H), 7.25(d, 1H), 7.18 (dd, 1H), 4.13 (q, 2H), 3.15 (t, 2H), 2.61 (t, 2H), 1.13(t, 3H).

(ii) 3-(2,4-Dichlorophenylthio)propanoic Acid

The sub-title compound was prepared using the method described inExample 1(ii) above, starting from 3-(2,4-dichlorophenylthio)propanoicacid, ethyl ester (5.8 g, 20.8 mmol; from step (i) above). Yield: 3.43 g(66%).

MS(m/z) 249 (M−1)⁻.

(iii) 6,8-Dichloro-4-thiochromanone

A deep red colour was immediately observed when3-(2,4-dichlorophenylthio)propanoic acid (1.20 g, 4.78 mmol; from step(ii) above) was added to conc. sulfuric acid (30 mL). After stirringovernight at ambient temperature the reaction mixture was poured intoice. The red colour disappeared. Extraction with diethyl ether, washingwith aq., sat. sodium hydrogencarbonate, drying (MgSO₄) and removal ofthe solvent in vacuo afforded a deep yellow solid.

Yield: 0.93 g (84%). ¹H NMR (400 MHz; CDCl₃) δ 8.01 (d, 1H), 7.47 (d,1H), 3.23 (m, 2H), 2.96 (m, 2H).

(iv) 6,8-Dichloro-4-hydroxythiochromane-4-yl-carboxylic Acid, EthylEster

The sub-title compound was prepared according to the method of C. F.Bigge et al. in J. Med. Chem., (1993), 36, 1977 using6,8-dichloro-4-thiochromanone (2.20 g, 9.44 mmol; from step (iii)above). Yield: 2.75 g (95%). ¹H NMR (500 MHz; CDCl₃) δ 7.30(d, 1H), 7.08(d, 1H), 4.24-4.35 (m, 2H), 3.23 (m, 1H), 3.04 (ddd, 1H), 2.29-2.41 (m,2H), 1.25 (t, 3H).

(v) 6,8-Dichloro-4-hydroxythiochromane-4-yl-carboxylic Acid

The sub-title compound was prepared using the method described inExample 1(v) above, starting from6,8-dichloro-4-hydroxychromane-4-yl-carboxylic acid, ethyl ester (2.74g, 8.92 mmol; from step (iv) above).

Yield: 2.17 g (87%). MS(m/z) 277 (M−1)⁻. ¹H NMR (400 MHz; CDCl₃) δ 7.34(d, 1H), 7.21 (d, 1H), 3.21 (m, 1H), 3.12 (ddd, 1H), 2.35-2.50 (m, 2H),2.13 (s, 1H).

(vi) (R)-6,8-Dichloro-4-hydroxythiochromane-4-yl-carboxylic Acid

The enantiomers of 6,8-dichloro-4-hydroxychromane-4-yl-carboxylic acid(2.17 g, 7.77 mmol; from step (v) above) were separated using chiralchromatography (Kromasil TBB; heptane: ethyl acetate: formic acid)affording 0.74 g of the faster moving, not wanted S-enantiomer, and 0.72g (66%) of the slower moving sub-title compound. ee=98.4%

(vii) (R)-6,8-Dichloro-4-hydroxythiochromane-4-yl-C(O)-Aze-Pab(Teoc)

The sub-title compound was prepared analogously to the method describedin Example 1(viii) above, starting from(R)-6,8-dichloro-4-hydroxychromane-4-yl-carboxylic acid (0.19 g, 0.68mmol; from step (vi) above) and H-Aze-Pab(Teoc) (0.37 g, 0.82 mmol;prepared as described in international patent application WO 98/57932).Yield: 0.28 g (65%). MS(m/z) 635 (M−1)⁻, 637 (M+1)⁺.

(viii)(R)-6,8-Dichloro-4-hydroxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-Pab(Teoc)

The sub-title compound was prepared analogously to the method describedin Example 1(viii) (alternative preparation, second step) above,starting from(R)-6,8-dichloro-4-hydroxy-thiochromane-4-yl-C(O)-Aze-Pab(Teoc) (0.100g, 0.157 mmol; from step (vii) above) and m-chloroperbenzoic acid (0.125g of 60%; 0.72 mmol). Yield: 0.040 g (38%). MS(m/z) 667 (M−1)⁻, 669(M+1)⁺.

(ix)(R)-6,8-Dichloro-4-hydroxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-PabxCF₃COOH

Trifluoroacetic acid (1.0 mL) was added to a stirred ice/water-cooledsolution of (R)-6,8-dichloro-4-hydroxy-1,1-dioxothiochromane-4-yl—C(O)-Aze-Pab(Teoc) (40mg, 60 μmol; from step (viii) above) in dichloromethane (10 mL). Thecooling bath was removed after 30 min. After 1 h at rt, acetonitrile (30mL) was added and the solvents were carefully removed under reducedpressure. The residue was dissolved in water and then freeze-dried.

Yield: 27 mg (68%). MS(m/z) 525 (M−1)⁻, 523 (M+1)⁺. ¹H NMR (400 MHz;CD₃OD): (complex due to diastereomers/rotamers) δ 8.74 (m, 1H),7.45-7.80 (several peaks, 6H), 5.53 (dd, 0.4H, rotamer), 4.35-4.88(several peaks, 3.8H), 4.09 (m, 0.4H, rotamer), 4.00 (m, 0.4H, rotamer),3.87 (m, 0.4H, rotamer), 3.68 (m, 1.2H, rotamer), 3.45 (ddd, 0.4H);2.78-2.90 (m, 1H), 2.73 (m, 0.4H, rotamer); 2.46-2.64 (several peaks,1.6H); 2.31 (m, 0.6H, rotamer); 2.15 (m, 0.4H, rotamer). ¹³C NMR (100MHz; CD₃OD): δ 174.2; 173.4; 168.2.

Example 3 (R)-6-Chloro-4-hydroxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-Pab

(i) 6-Chloro-4-hydroxythiochromane-4-yl-carboxylic Acid, Methyl Ester

The sub-title compound was prepared according to the method of C. F.Bigge et al. in J. Med. Chem., (1993), 36, 1977 from6-chloro-4-thiochromanone (1.74 g, 8.76 mmol) using methanol instead ofethanol.

Yield: 0.625 g (28%). ¹H NMR (500 MHz; CDCl₃) δ 7.07-7.15 (severalpeaks, 3H), 3.93 (s, 1H), 3.93 (s, 3H), 3.23 (ddd, 1H), 2.99 (ddd, 1H),2.42 (ddd, 1H), 2.34 (ddd, 1H).

(ii) 6-Chloro-4-hydroxythiochromane-4-yl-carboxamide

The sub-title compound was obtained as the main product in the previousstep and isolated using reversed phase HPLC (acetonitrile: 0.1 M aq.ammonium acetate).

Yield: 1.49 g (70%). ¹H NMR (500 MHz; CDCl₃) δ 7.35 (d, 1H), 7.15 (dd,1H), 7.09 (d, 1H), 6.32 (bs, 1H), 5.90 (bs, 1H), 3.86 (bs, 1H), 3.19(ddd, 1H), 3.03 (ddd, 1H), 2.40 (ddd, 1H), 2.30 (ddd, 1H).

(iii) 6-Chloro-4-hydroxythiochromane-4-yl-carboxylic Acid

Potassium hydroxide (5.1 g, 91 mmol) and6-chloro-4-hydroxythiochromane-4-yl-carboxamide (1.36 g, 5.58 mmol; fromstep (ii) above) were dissolved in water (25 mL) and isopropanol andrefluxed for 2 d. The isopropanol was removed under reduced pressure andthe aqueous residue was washed with diethyl ether. Acidification withaq. HCl, extraction with ethyl acetate, and then drying andconcentration in vacuo afforded the sub-title compound. Yield: 0.87 g(64%).

MS(m/z) 243 (M−1)⁻. Alternative method: The sub-title compound was alsoobtained using the method in Example 1(v) above, starting from6-chloro-4-hydroxythiochromane-4-yl-carboxylic acid, methyl ester (0.63g, 2.4 mmol; from step (i) above). Yield: 0.55 g (92%).

(iv) (R)-6-Chloro-4-hydroxythiochromane-4-yl-carboxylic Acid

The enantiomers of 6-chloro-4-hydroxythiochromane-4-yl-carboxylic acid(1.4 g, 5.7 mmol; from step (iii) above) were separated using chiralchromatography (Kromasil TBB; heptane: ethyl acetate: formic acid)yielding 0.500 g (71%) of the slower moving sub-title compound.

(v) (R)-6-Chloro-4-hydroxychromane-4-yl-C(O)-Aze-Pab(Teoc)

The sub-title compound was prepared analogously to the method describedin Example 1(viii) (alternative preparation, first step) above, startingfrom (R)-6-chloro-4-hydroxychromane-4-yl-carboxylic acid (0.178 g, 0.727mmol; from step (iv) above) and H-Aze-Pab(Teoc) (0.36 g, 0.80 mmol;prepared as described in international patent application WO 98/57932).Yield: 0.31 g (71%). MS(m/z) 601 (M−1)⁻; 603 (M+1)⁺.

(vi)(R)-6-Chloro-4-hydroxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-Pab(Teoc)

The sub-title compound was prepared analogously to the method describedin Example 1(viii) (alternative preparation, second step) above,starting from(R)-6-chloro-4-hydroxythio-chromane-4-yl-C(O)-Aze-Pab(Teoc) (0.113 g,0.187 mmol; from step (v) above) and m-chloroperbenzoic acid (0.16 g of˜60%, 0.56 mmol). Yield: 0.029 g (27%). MS(m/z) 633 (M−1)⁻, 635 (M+1)⁺.

(vii)(R)-6-Chloro-4-hydroxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-PabxHOAc

The title compound was prepared analogously to the method described inExample 1(ix) above, starting from(R)-6-chloro-4-hydroxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-Pab(Teoc)(0.029 g, 0.046 mmol; from step (vi) above) and tetrabutylammoniumfluoride (0.080 mL of 1.0 M, 0.080 mmol).

Yield: 0.023 g (91%). MS(m/z) 489 (M−1)⁻, 491 (M+1)⁺. ¹H NMR (400 MHz;CD₃OD): (complex due to diastereomers/rotamers) δ 7.47-7.84 (severalpeaks, 7H); 5.55 (dd, 0.3H, rotamer); 4.43-4.66 (several peaks, 2.7H);4.30 (m, 0.7H, rotamer); 4.09 (m, 0.5H, rotamer); 4.00 (m, 0.5H,rotamer); 3.77 (ddd, 0.5H, rotamer); 3.54-3.67 (several peaks, 1.3H);3.43 (ddd, 0.5H, rotamer); 2.52-2.99 (several peaks, 3H); 2.24-2.33 (m,1H); 2.10-2.20 (m, 0.7H), rotamer), 1.92 (s, 3H), 1.75-1.85 (m, 0.3H),rotamer).

¹³C NMR (100 MHz; CD₃OD): (complex due to diastereomers/rotamers,carbonyl and/or amidine carbons) δ 173.5; 172.9; 168.1.

Example 4(R)-6-Chloro-4-hydroxy-8-methoxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-Pab(OBn)

(i)(R)-6-Chloro-4-hydroxy-8-methoxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-Pab(OBn)(Teoc)

(R)-6-Chloro-4-hydroxy-8-methoxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-Pab-(Teoc)(0.040 g, 0.060 mmol; see Example l(viii) above) andO-benzylhydroxylamine×HCl (0.047 g, 0.29 mmol) in THF (15 mL) wereheated to 60° C. for 2d. After concentration under reduced pressure, theresidue was dissolved in ethyl acetate and washed with water and brine.Drying (MgSO₄) and removal of the solvent in vacuo afforded a colourlesssolid residue. Yield: 0.046 g (99%). MS(m/z) 771 (M+1)⁺, 793 (M+23)⁺.

(ii)(R)-6-Chloro-4-hydroxy-8-methoxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-Pab(OBn)

A solution of(R)-6-chloro-4-hydroxy-8-methoxy-1,1-doxothjochromane-4-yl-C(O)-Aze-Pab(OBn)(Teoc)(0.046 g, 0.060 mmol; from step (i) above) and tetrabutylammoniumfluoride (0.20 mL of 120 M, 0.20 mmol) in acetonitrile (3 mL) was heatedto 60° C. for 3 h. After concentration under reduced pressure, theresidue was dissolved in ethyl acetate and washed with water and brine.Evaporation of the solvent in vacuo afforded only 0.019 g. The aqueousfractions were freeze dried, combined with the evaporation residue fromthe organic phase and purified using reversed-phase HPLC (acetonitrile:0.1 M aq. ammonium acetate) to afford, after freeze drying theappropriate fractions, the title compound as a colourless solid.

Yield: 0.00 9 g (24%). MS(m/z) 625 (M−1)⁻, 627 (M+1)⁺. ¹H NMR (400 MHz;CDCl₃): δ 7.59 (d, 2H), 7.23-7.45 (several peaks, is 7H), 6.99 (d, 1H),6.81 (d, 1H), 5.12 (s, 2H); 4.90 (dd, 1H), 4.85 (bs, 2H), 4.40-4.54(AB-part of ABX-system, 2H), 4.09 (m, 1H), 3.97 (s, 3H), 3.84 (ddd, 1H),3.28 (ddd, 1H), 3.09 (m, 1H), 2.79 (ddd, 1H), 2.63 (m, 1H), 2.32 (m,1H), 2.21 (m, 1H). ¹³C NMR (100 MHz; CDCl₃): (carbonyl and/or amidinecarbons) δ 169.1, 158.3, 151.6.

Example 5(R)-6-Chloro-4-hydroxy-8-methoxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-Pab(C(O)-O-cyclopentyl)

Sodium hydroxide (aq.; 0.5 B of 2.0 M, 1.0 9mol) was added to a solutionof(R)-6-chloro-4-hydroxy-8-methoxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-PabxHOAC(21 mg, 40 μmol; see Example 1 above) and cyclopentyl chloroformate (11mg, 74 μmol) in dichloromethane, and the mixture was stirred at rt for 3h. After dilution with water, the resultant mixture was extracted withdichloromethane. The combined organic fractions were concentrated invacuo and purified using flash chromatography (SiO₂; dichloromethane,then with EtOAc/MeOH 95/5). The fractions of interest were concentratedunder reduced pressure, then dissolved in water/acetonitrile and freezedried to afford the title compound. Yield: 16 mg (60%).

MS(m/z) 631 (M−1)⁻, 633 (M+1)⁺. ¹H NMR (300 MHz; CDCl₃): (minor rotamer(˜10%) not reported) δ 7.75 (d, 2H), 7.49 (t, 1H), 7.23 (d, 2H), 6.92(d, 1H), 6.85 (d, 1H), 5.14 (m, 1H); 4.82 (broad dd, 2H), 4.34-4.58(AB-part of ABX-system, 2H), 4.15 (m, 1H), 3.97 (bs, 1H), 3.92 (s, 3H),3.71 (ddd, 1H), 3.39 (ddd, 1H), 3.24 (m, 1H), 2.79 (ddd, 1H), 2.56 (m,1H), 2.22-2.38 (several peaks, 2H), 1.51-2.00 (several peaks, 8H). ¹³ CNMR (100 MHz; CDCl₃): (carbonyl and/or amidine carbons) δ 173.1, 171.1,169.3, 158.2.

Example 6(R)-6,8-Dichloro-4-hydroxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-Pab(OMe)

(i)(R)-6,8-Dichloro-4-hydroxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-Pab(OMe)(Teoc)

(R)-6,8-Dichloro-4-hydroxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-Pab(Teoc)(0.046 g, 0.069 mmol; from Example 2(viii) above) andO-methyl-hydroxylamine×HCl (0.043 g, 0.51 mmol) in THF (5 mL) wererefluxed overnight. After concentration under reduced pressure, theresidue was dissolved in ethyl acetate and washed with water and brine.Drying (MgSO₄) and removal of the solvent in vacuo afforded a colourlesssolid residue. Yield: 0.042 g (87%).

(ii)(R)-6,8-Dchloro-4-hydroxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-Pab(OMe)

Trifluoroacetic acid (1.0 mL) was added to a stirred ice/water-cooledsolution of(R)-6,8-dichloro-4-hydroxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-Pab(OMe)(Teoc)(0.042 g, 0.060 mmol; from step (i) above) in dichloromethane (3 mL).The cooling bath was removed after 1 h. After 3 h at rt, acetonitrilewas added and the solvents were carefully removed under reducedpressure. The crude product was purified using reversed-phase HPLC(acetonitrile: 0.1 M aq. ammonium acetate) to afford, after freezedrying the appropriate fractions, the title compound as a colourlesssolid.

Yield: 0.018 g (54%). MS(m/z) 553 (M−1)⁻, 555 (M+1)⁺. ¹H NMR (400 MHz;CDC)₃): (minor rotamer (˜10%) not reported) δ 7.57 (d, 2H), 7.51 (d,1H), 7.10-7.55 (several peaks, 4H), 4.96 (bs, 2H); 4.94 (dd, 1H),4.38-4.54 (AB-part of ABX-system, 2H), 4.18 (m, 1H), 3.91 (s, 3H), 3.76(ddd, 1H), 3.43 (ddd, 1H), 3.23 (m, 1H), 2.84 (ddd, 1H), 2.58 (m, 1H),2.27-2.43 (several peaks, 2H). ¹³C NMR (100 MHz; CDCl₃): (carbonyland/or amidine carbons) δ 172.7, 169.1, 151.5.

Example 76-Chloro-8-difluoromethoxy-4-hydroxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-Pab

(i) 6-Chloro-8-hydroxy-4-thiochromanone, Ethylene Ketal

To a solution of 6-chloro-8-methoxy-4-thiochromanone (7.64 g, 33.4 mmol;see Example l(iii) above) in benzene (300 mL) under nitrogen was addedethylene glycol (9.34 mL, 167 mmol) and PTSA·H₂O (catalytic amount). Themixture was heated to reflux with a Dean-Stark trap for 48 h. In orderto drive the reaction to completion it was necessary to add molecularsieves (type 4 Å, 8-12 mesh) to the trap. The reaction was cooled toroom temperature, solid NaHCO₃ (0.10 g) was added and the mixture wasdiluted with EtOAc (200 mL). The organic layer was washed with saturatedNaHCO₃ (2×150 mL), brine (150 mL), dried (Na₂SO₄), filtered, andconcentrated in vacuo to afford 9.03 g (ca. 99%) of the intermediateketal as a crude yellow solid. Ethanethiol (24.7 mL, 334 mmol) was addedto a mixture of the above ketal (13.0 g, 47.7 mmol) in DMF (250 mL)under nitrogen and cooled to 0° C. Sodium hydride (8.0 g, 334 mmol) wasadded slowly and the reaction stirred at 0° C. for 15 minutes, thenplaced in an oil bath and heated to 135° C. for 1.5 h. Once the reactionhad cooled to room temperature, it was poured into an ice-water mixture(300 g) containing saturated NH4Cl solution (70 mL). The aqueous layerwas extracted with EtOAc (2×150 mL) and the combined organic extractswere washed with brine (200 mL), dried (Na₂SO₄), filtered, andconcentrated in vacuo. The brown residue was chromatographed on silicagel eluting with CH₂Cl₂ to afford 11.3 g (92%) of the sub-title compoundas a yellow solid.

¹H NMR (300 MHz, CD₃OD) δ 7.05 (s, 1H), 6.68 (s, 1H), 4.05-4.20 (m, 4H),3.07 (t, J =8.0 Hz, 2H), 2.13 (t, J=8.0 Hz, 2H).

(ii) 6-Chloro-8-difluoromethoxy-4-thiochromanone, Ethylene Ketal

A solution of 6-chloro-8-hydroxy-4-thiochromanone, ethylene ketal (6.0g, 23.2 mmol; from step (i) above) in 2-propanol (100 mL) and 30% KOH(50 mL) was heated to reflux and Freon 22 was bubbled directly into thereaction for 50 min. at <2.0 psi. The mixture was cooled to roomtemperature, partitioned between CH₂Cl₂ and H₂O (2:1, 450 mL) and theaqueous layer was extracted with CH₂Cl₂ (150 mL). The combined organiclayers were washed with brine (150 mL), dried (Na₂SO₄), filtered, andconcentrated in vacuo. The resulting residue was chromatographed onsilica gel eluting with CH₂Cl₂ to afford 7.2 g (78%) of the sub-titlecompound as a yellow solid.

is ¹H NMR (300 MHz, CDCl₃) δ 7.45 (s, 1H), 7.10 (s, 1H), 6.45 (t, J=74Hz, 1H), 4.09-4.25 (m, 4H), 3.17 (t, J=7.5 Hz, 2H), 2.17 (t, J=7.5 Hz,2H).

(iii) 6-Chloro-8-difluoromethoxy-4-thiochromanone

A solution of 6-chloro-8-difluoromethoxy-4-thiochromanone, ethyleneketal (5.11 g, 16.6 mmol; from step (ii) above) in THF (105 mL) and 0.5N HCl (70 mL) was stirred at room temperature for 96 h. The reaction wasneutralized with saturate d NaHCO₃ (150 mL) and extracted with CH₂Cl₂(2×100 mL). The combined organic extracts were washed with brine (100mL), dried (Na₂SO₄), filtered, and concentrated in vacuo to afford 4.40g (ca. 100%) of the sub-title compound as a yellow solid which was usedwithout further purification.

¹H NMR (300 MHz, CDCl₃) δ 8.00 (s, 1H), 7.30 (s, 1H), 6.56 (t, J=74 Hz,1H), 3.24 (t, J=7.5 Hz, 2H), 3.00 (t, J=7.5 Hz, 2H).

(iv) 6-Chloro-8-difluoromethoxy-4-methylenethiochromane

A solution of 6-chloro-8-difluoromethoxy-4-thiochromanone (2.5 g, 9.40mmol; from step (iii) above) in anhydrous Et₂O (150 mL) was cooled to 0°C. under nitrogen. Tebbe Reagent (21.0 mL of 0.5M in toluene, 10.3 mmol)was added dropwise via syringe, and the mixture was stirred at 0° C. for2 h and then stirred at room temperature for 1 h. The reaction wascooled to 0° C. and slowly quenched with MeOH (ca. 50 mL) until thesolution solidified. The mixture was diluted with EtOAc/H₂₀ (4:1, 100mL) and filtered through a pad of Celite. The Celite was washed withEtOAc (800 mL) and the filtrate was concentrated in vacuo. The crudedark red oil was chromatographed on silica gel eluting with Hex:EtOAc(4:1) to afford 0.88 g (36%) of sub-title compound as a dark yellow oil.¹H NMR (300 MHz, CDCl₃) δ 7.40 (s, 1H), 7.05 (s, 1H), 6.46 (t, J=75 Hz,1H), 5.53 (s, 1H), 5.10 (s, 1H), 3.06 (t, J=7.5 Hz, 2H), 2.80 (t, J=7.5Hz, 2H).

(v) (R)-6-Chloro-8-difluoromethoxy-4-hydroxy-4-hydroxymethylthiochromane

2-Methyl-2-propanol (120 mL), H₂O (120 mL), and AD-mix-β (13.3 g) werecombined together and cooled to 0° C. Concurrently,6-chloro-8-difluoromethoxy-4-methylenethiochromane (2.65 g, 10.1 mmol;from step (iv) above) was cooled to −5° C. in an ice/MeOH bath. Oncesufficiently cooled, the AD-mix-β solution was added and stirred at <0°C. for 2 h, then at room temperature for 4 h. The reaction was quenchedwith solid sodium sulfite (ca. 5.0 g) resulting in a clear solutionwhich was then partitioned between EtOAc/H₂O (1:1, 300 mL) and furtherextracted with EtOAc (100 mL). The organic extracts were combined andwashed with 5 M H₂SO₄ (75 mL), saturated NaHCO₃ (150 mL), and brine (150mL), dried (Na₂SO₄), filtered, and concentrated in vacuo to afford 2.99g (ca. 100%) of the sub-title compound as a yellow solid which was usedwithout further purification. ¹H NMR (300 MHz, CD₃OD) δ 7.55 (s, 1H),7.08 (s, 1H), 6.78 (t, J=75 Hz, 1H), 3.70 (d, J=12 Hz, 1H), 3.50 (d,J=12 Hz, 1H), 2.97-3.20 (m, 2H), 2.46-2.55 (m, 1H), 1.91-2.02 (m, 1H).

(vi)(R)-6-Chloro-8-difluoromethoxy-4-hydroxythiochromane-4-yl-carboxylicAcid

Sodium carbonate (0.72 g, 6.7 mmol) and 5% Pt/C (1.50 g, 75% weight ofreactant) were added to a solution of(R)-6-chloro-8-difluoromethoxy-4-hydroxy-4-hydroxymethylthiochromane(2.0 g, 6.7 mmol; from step (v) above) in H₂O (180 mL). The mixture wasvigorously stirred at ca. 95° C. for 20-40 h with air being directlybubbled into the reaction mixture. The reaction was cooled to roomtemperature and filtered through a pad of Celite with saturated Na₂CO₃solution (ca. 100 mL). The resulting liquid was slowly acidified with 2N HCl (ca. 200 mL) and extracted with EtOAc (2×100 mL). The combinedorganic layers were washed with brine (75 mL), dried (Na₂SO₄), filtered,and concentrated in vacuo to afford 1.56 g (ca. 75%) of the sub-titlecompound as a brown oil which was used without further purification.

¹H NMR (300 MHz, CD₃OD) δ 7.24 (s, 1H), 7.12 (s, 1H), 6.81 (t, J=75 Hz,1H), 3.04-3.19 (m, 2H), 2.40-2.50 (m, 1H), 2.25-2.35 (m, 1H).

(vii)(R)-6-Chloro-8-difluoromethoxy-4-hydroxy-1,1-dioxothiochromane-4-yl-carboxylicAcid

A mixture of(R)-6-chloro-8-difluoromethoxy-4-hydroxythiochromane-4-yl-carboxylicacid (0.50 g, 1.6 mmol; from step (vi) above), wet alumina (prepared byadding H₂O (1 mL) to alumina (5.0 g) and shaking until a free flowingpowder was obtained, ca. 0.82 g, 8.0 mmol), and Oxone® (4.9 g, 8.0 mmol)in CHCl₃ (19 mL) was heated to 40-45° C. for 20 h. The reaction wascooled to room temperature, filtered through a Buichner funnel withEtOAc (50 mL), and concentrated in vacuo to afford 0.54 g (98%) of thesub-title compound as a brown foam which was used without furtherpurification.

¹H NMR (300 MHz, CD₃OD) δ 7.44 (s, 2H), 6.94 (t, J=75 Hz, 1H), 3.61-3.75(m, 2H), 2.81-2.91 (m, 1H), 2.52-2.62 (m, 1H).

(viii)(R)-6-Chloro-8-difluoromethoxy-4-hydroxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-Pab(Teoc)

To a solution of(R)-6-chloro-8-difluoromethoxy-4-hydroxy-1,1-dioxothiochromane-4-yl-carboxylicacid (0.54 g, 1.6 mmol; from step (vii) above) in DMF (20 mL) underargon at 0° C. was added H-Aze-Pab(Teoc)×HCl (0.92 g, 2.1 mmol), PyBOP(0.91 g, 1.7 mmol), and Hünig's Base (0.7 mL, 4.0 mmol). The reactionwas stirred at 0° C. for 2 h and then at room temperature for 20 h. Themixture was concentrated under reduced pressure and the residue waschromatographed twice on silica gel, eluting first with CHCl₃:EtOH(10:1) and then with EtOAc:EtOH (10:1) to afford 0.44 g (40%) of thesub-title compound as a yellow crystalline solid.

mp 135-145° C. ¹H NMR (300 MHz, CD₃OD, complex mixture of rotamers) δ7.74-7.82 (m, 2H), 7.36-7.59 (m, 4H), 6.81 (t, J=75 Hz, 1H), 5.48-5.55and 4.80-4.89 (m, 1H), 4.71-3.39 (m, 8H), 2.90-2.10 (m, 4H), 1.08 (m,2H), 0.10 (s, 9H). APCI-MS(m/z) 701 (M+1)⁺.

(ix)(R)-6-Chloro-8-difluoromethoxy-4-hydroxy-1,1-dioxothiochromane-4-yl-C(O)-Aze-PabxHOAc

Trifluoroacetic acid (3.0 mL) was added to a stirred ice/water-cooledsolution of(R)-6-cloro-8-difluoromethoxy-4-hydroxy-1,1-dioxothiochromane-4-yl-Aze-Pab(Teoc)(71 mg; 0.10 mmol; from step (viii) above) in methylene chloride (1 mL).After 1 h methylene chloride (3 mL) was added and the solvents werecarefully removed under reduced pressure. The residue was dissolved inwater/acetonitrile and freeze dried. Purification with reversed-phaseHPLC (0.1M aq. ammonium acetate/acetonitrile, 7:3) afforded the titlecompound as a white solid.

Yield: 45 mg (72%). APCI-MS(m/z) 557 (M+1)⁺, 555 (M−1)⁻. ¹H NMR (400MHz; CD₃OD): δ 7.77 (complex due to diastereomers/rotamers) (d, 0.8H,rotamer); 7.68 (d, 1.2H, rotamer); 7.58 (d, 0.6H, rotamer); 7.55 (d,0.8H, rotamer); 7.48 (d, 1.2H, rotamer); 7.47 (d, 0.4H, rotamer); 7.40(m, 0.4H, rotamer); 7.38 (m, 0.6H, rotamer); 6.90 (t, 1H); 5.54 (dd,0.4H, rotamer); 4.68 (m, 0.6H, rotamer); 4.35-4.61 (several peaks, 3H);4.09 (m, 0.4H, rotamer); 3.99 (m, 0.6H, rotamer); 3.82 (apparent dt,0.4H, rotamer); 3.65 (apparent t, 1.2H, rotamer); 3.44 (m, 0.4H,rotamer); 2.69-2.90 (several peaks, 1.4H); 2.48-2.64 (several peaks,1.6H); 2.29 (m, 0.6H, rotamer); 2.15 (m, 0.4H, rotamer); 1.89 (s, 3H).

³C NMR (75 MHz; CD₃OD): (complex due to diastereomers/rotamers, carbonyland/or amidine carbons) 174.1; 173.4; 173.3; 172.9; 168.1.

Example 8 6-Chloro-4-(R)-hydroxy-8-methoxy-1-(R orS)-oxo-thiochromane-4-yl-C(O)-Aze-PabxTFA

(i) 6-Chloro-4-(R)-hydroxy-8-methoxy-1-(R orS)-oxo-thiochromane-4-yl-carboxylic acid (I) and6-Chloro-4-(R)-hydroxy-8-methoxy-1-(S orR)-oxo-thiochromane-4-yl-carboxylic Acid (II)

A mixture of(R)-6-chloro-4-hydroxy-8-methoxythiochromane-4-yl-carboxylic acid (700mg, 2.60 mmol, see Example 1(vi) above) and 35% H₂O₂ (3.4 mL, 30.1 mmol)in CH₂Cl₂ (40 mL) was stirred at 0° C. for 2 h, and then overnight at25° C. under nitrogen. The mixture was concentrated in vacuo andchromatographed on silica gel eluting with CHCl₃:MeOH:concentrated NH₄OH(7.5:2.0:0.5) to afford 320 mg of the ammonium salt of the sub-titlecompound diastereomer (I) and 190 mg of the ammonium salt of thesub-title compound diastereomer (II). Both materials were eachseparately dissolved in H₂O (40 mL), acidified with 1N HCl, andextracted with EtOAc (2×50 mL). The organic extracts of each were dried(Na₂SO₄), filtered, and concentrated to afford 290 mg (40%) of sub-titlecompound (I) and 170 mg (22%) of sub-title compound (II), respectively.

For sub-title compound diastereomer (I): ¹H NMR (300 MHz, CD₃OD) 7.25(s, 1H), 7.05 (s, 1H), 4.00 (s, 3H), 3.60-3.68 (m, 1H), 3.20-3.28 (m,1H), 2.80-2.92 (m, 1H), 2.46-2.58 (m, 1H) APCI-MS: (M+1)=291 m/z

For sub-title compound diastereomer (II):

¹H NMR (300 MHz, CD₃OD) 7.35 (s, 1H), 7.20 (s, 1H), 4.00 (s, 3H),3.42-3.58 (m, 1H), 3.18-3.30 (m, 1H), 2.70-2.88 (m, 1H), 2.30-2.45 (m,1H). APCI-MS: (M+1)=291 m/z

(ii) 6-Chloro-4-(R)-hydroxy-8-methoxy-1-(R orS)-oxo-thiochromane-4-yl-C(O)-Aze-Pab(Teoc)

To a solution of 6-chloro-4-(R)-hydroxy-8-methoxy-1-(R orS)-oxo-thiochromane-4-yl-carboxylic acid (diastereomer (I) from step (i)above, 284 mg, 0.98 mmol) in DMF (10 mL) under nitrogen was addedH-Aze-Pab(Teoc)×HCl (571 mg, 1.27 mmol), PyBOP (559 mg, 1.07 mmol), andDIPEA (315 mg, 2.44 mmol) at 0° C. The reaction was stirred at 0° C. for2 h and then at room temperature overnight. The mixture was concentratedin vacuo and the residue chromatographed twice on silica gel, elutingtwice with CHCl₃:EtOH (9:1) to afford 190 mg (30%) of the sub-titlecompound as a white solid.

mp: 225-230° C. R_(f)=0.57 (9:1 CHCl₃:EtOH) ¹H NMR (300 MHz, CD₃OD/CDCl₃(1:1), complex mixture of rotamers) 7.83 (d, J=8 Hz, 2H), 7.38 (d, J=7Hz, 2H), 7.08 (s, 1H), 6.99 (s, 1H), 4.87-4.92 (m, 1H), 4.51-4.54 (m,2H), 4.21-4.30 (m, 4H), 4.00 (s, 3H), 3.12-3.39 (m, 4H), 2.39-2.50 (m,1H), 2.16-2.22 (m, 1H), 1.08-1.14 (m, 2H), 0.08 (s, 9H) APCI-MS:(M+1)=649 m/z.

(iii) 6-Chloro-4-(R)-hydroxy-8-methoxy-1-(R orS)-oxo-thiochromane-4-yl-C(O)-Aze-PabxTFA

To a solution of 6-chloro-4-(R)-hydroxy-8-methoxy-1-(R orS)-oxo-thiochromane-4-yl-C(O)-Aze-Pab(Teoc) (90 mg, 0.14 mmol, from step(ii) above) in dichloromethane (2 mL), trifluoroacetic acid (2 mL) wasadded. The solution was left at ambient temperature for 1 h whereupon itwas concentrated under reduced pressure. The residue was dissolved indeionized water and freeze dried. Yield: 80 mg (0.13 mmol).

¹H-NMR (500 MHz; CD₃OD): (complex mixture due to rotamers) 8.75 (t,0.75H, rotamer); 8.63 (t, 0.25H, rotamer); 7.76 (d, 0.5H, rotamer); 7.72(d, 1.5H, rotamer); 7.55 (d, 2H, mix of rotamers); 7.23 (s, 1H, mix ofrotamers); 7.11 (s, 0.75H, rotamer); 7.01 (s, 0.25H, rotamers); 5.45 (m,0.5H); 4.61 (m, 1H); 4.47 (m, 2H); 4.13 (dm, 0.5H); 3.98 (s, 3H); 3.83(q, 1H); 3.23 (m, 1.5H); 3.10 (m, 1.25H); 2.97 (m, 0.25H); 2.78 (m,0.25H); 2.49 (m, 0.75H); 2.26 (m, 1H); 2.18 (m, 1H) MS (m/z) 505 (M+1)⁺,503 (M-1)⁻.

Example 9 6-Chloro-4-(R)-hydroxy-8-methoxy-1-(S orR)-oxo-thiochromane-4-yl-C(O)-Aze-PabxTFA

(i) 6-Chloro-4-(R)-hydroxy-8-methoxy-1-(S orR)-oxo-thiochromane-4-yl-C(O)-Aze-Pab(Teoc)

To a solution of 6-chloro-4-(R)-hydroxy-8-methoxy-1-(S orR)-oxo-thiochromane-4-yl-carboxylic acid (diastereomer (II) from Example8(i) above, 166 mg, 0.57 mmol) in DMF (10 mL) under nitrogen were addedH-Aze-Pab(Teoc)×HCl (333 mg, 0.74 mmol), PyBOP (327 mg, 0.63 mmol), andDIPEA (184 mg, 1.43 mmol) at 0° C. The reaction was stirred at 0° C. for2 h and then at room temperature overnight. The mixture was concentratedin vacuo and the residue chromatographed twice on silica gel, elutingfirst with CHCl₃:EtOH (9:1) and second with CHCl₃:MeOH (20:1) to afford80 mg (22%) of the sub-title compound as a white solid.

mp: 140-145° C. R_(f)=0.60 (9:1 CHCl₃:EtOH) ¹H NMR (300 MHz, CD₃OD,complex mixture of rotamers) 7.80 and 7.72 (d, J=8 Hz, 2H), 7.42-7.49(m, 2H), 7.32 (s, 1H), 7.17 (s, 1H), 5.52-5.57 (m, 1H), 4.44-4.91 (m,4H), 4.19-4.25 (m, 2H), 3.97 (s, 3H), 3.00-3.72 (m, 3H), 2.10-2.82 (m,3H), 1.04-1.11 (m, 2H), 0.07 (s, 9H) APCI-MS: (M+1)=649 m/z.

(ii) 6-Chloro-(R)-4-(R)-hydroxy-8-methoxy-1-(S orR)-oxo-thiochromane-4-yl-C(O)-Aze-PabxTFA

To a solution of 6-chloro-4-(R)-hydroxy-8-methoxy-1-(S orR)-oxo-thiochromane-4-yl-C(O)-Aze-Pab(Teoc) (35 mg, 0.054 mmol from step(i) above) in dichloromethane (2 mL), was added trifluoroacetic acid (2mL). The solution was left at ambient temperature for 1 h whereupon itwas concentrated at reduced pressure. The residue was dissolved indeionized water and freeze dried. Yield: 30 mg (0.048 mmol).

¹H-NMR (500 MHz; CD₃OD): (complex mixture due to rotamers) 548.82 (m,0.2H); 8.65 (m, 0.2H); 7.77 (d, 0.9H); 7.66 (d, 0.8H); 7.59 (d, 0.9H);7.43 (d, 0.8H); 7.40 (d, 0.4H); 7.30 (d, 0.4H); 7.16 (dd, 0.8H); 5.55(m, 0.45H); 4.70 (m, 0.6H); 4.59 (m, 0.6H); 4.51 (m, 1.8H); 4.05 (q,0.45H); 3.97 (s, 3H); 3.94 (m, 0.4H); 3.58 (m, 0.4H); 3.38 (m, 0.4H);3.16 (m, 0.4H); 3.03 (m, 0.4H); 2.75 (m, 0.8H); 2.58 (m, 0.4H); 2.45 (m,1.2H); 2.30 (m, 0.4H); 2.15 (m, 0.45H) MS (m/z): 505 (M+1)⁺, 503 (M−1)⁻.

Example 10

Title compounds of Examples 1, 2, 3, 7, 8 and 9 were tested in Test Aabove and were found to exhibit an IC₅₀TT value of less than 0.05 μM.

Example 11

Title compounds of Examples 4, 5 and 6 are tested in Test E above andare found to exhibit oral and/or parenteral bioavailability in the ratas the corresponding active inhibitor (free amidine).

Example 12

Title compounds of Examples 4, 5 and 6 are tested in Test G above andare found to exhibit oral and/or parenteral bioavailability in the ratas the corresponding active inhibitor (free amidine).

Abbreviations Ac = acetyl AcOH = acetic acid API = atmospheric pressureionisation (in relation to MS) aq. = aqueous AUC = area under the curveAze = (S)-azetidine-2-carboxylate (unless otherwise specified) AzeOH =azetidine-2-carboxylic acid Bn = benzyl BSA = bovine serum albumin CI =chemical ionisation (in relation to MS) d = day(s) DCC = dicyclohexylcarbodiimide DIPEA = diisopropylethylamine DMAP = 4-(N,N-dimethyl amino)pyridine DMF = dimethylformamide DMSO = dimethylsulfoxide EDC =1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride Et = ethylether = diethyl ether EtOAc = ethyl acetate EtOH = ethanol h = hour(s)HATU = O-(azabenzotriazol-1-yl)-N,N,N′,N′- tetramethyluroniumhexafluorophosphate HBTU =[N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium hexafluorophosphate]HCl = hydrochloric acid, hydrogen chloride gas or hydrochloride salt(depending on context) Hex = hexanes HOAc = acetic acid HPLC = highperformance liquid chromatography LC = liquid chromatography Me = methylMeOH = methanol min. = minutes MS = mass spectroscopy MTBE = methyltert-butyl ether NADH = nicotinamide adenine dinucleotide, reduced formNADPH = nicotinamide adenine dinucleotide phosphate, reduced form NIH =National Institute of Health (US) NIHU = National Institute of Healthunits Pab = para-amidinobenzylamino H-Pab = para-amidinobenzylamine PTSA= para-toluenesulphonic acid PyBOP =(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate RPLC= reverse phase high performance liquid chromatography rt = roomtemperature SOPs = standard operating procedures TBTU =[N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate]TEA = triethylamine Teoc = 2-(trimethylsilyl)ethoxycarbonyl TFA =trifluoroacetic acid THF = tetrahydrofuran THP = tetrahydropyranyl TLC =thin layer chromatography TMSCN = trimethylsilyl cyanide Z =benzyloxycarbonyl

Prefixes n, s, i and t have their usual meanings: normal, secondary, isoand tertiary.

What is claimed is:
 1. A compound of formula I,

wherein Y represents S(O) or S(O)₂; R¹ represents halo; and R²represents H, halo or C₁₋₄ alkoxy which latter group is optionallysubstituted by one or more halo groups; or a pharmaceutically acceptablesalt or prodrug thereof.
 2. A compound as claimed in claim 1, wherein R¹represents chloro.
 3. A compound as claimed in claim 1, wherein R²represents H, halo or C₁₋₂ alkoxy which latter group is optionallysubstituted by one or more halo groups.
 4. A compound as claimed inclaim 1, wherein R¹ represents chloro and R² represents H, chloro, OCH₃,OCHF₂ or OCF₃.
 5. A compound as claimed in claim 4, wherein R²represents OCH₃. 6.(R)-6-Chloro-4-hydroxy-8-methoxy-1,1-dioxothiochromane-4-yl-C(O)-(S)-Aze-Pabor a pharmaceutically acceptable salt or prodrug thereof.
 7. A processfor the preparation of a compound as claimed in claim 1, whichcomprises: (i) the coupling of a compound of formula III,

 wherein Y, R¹ and R² are as defined in claim 1, with4-amidinobenzyl-2-azetidinecarboxamide; (ii) the coupling of a compoundof formula IV,

 wherein Y, R¹ and R² are as defined in claim 1, withpara-amidino-benzylamine; (iii) complete or partial oxidation of acorresponding compound of formula V,

 wherein R¹ and R² are as defined in claim 1; (iv) deprotection of aprotected compound of formula I as defined in claim 1; or (v)introduction or interconversion of a substituent on the aromatic ring ina compound of formula I as defined in claim
 1. 8. A pharmaceuticallyacceptable compound of formula IA,

wherein Y, R¹ and R² are as defined in claim 1; D¹ and D² independentlyrepresent H, —OR⁷ or R⁸, or D¹, D² and R³ together with the amidinegroup to which they are attached, form a cyclic group of formula IIa,IIb, IIc, IId or IIe,

wherein wavy lines indicate the points of attachment to the benzenering; R³ represents H, or R³, D¹, and D² together with the amidine groupto which they are attached, form a cyclic group of formula Ia, IIb, IIc,IId or IIe; R⁴ and R⁵ independently represent H or C₁₋₄ alkyl; R⁶represents H, C₁₋₆ alkyl which latter group is optionally substituted byone or more halo groups or C(O)OR¹²; R⁷ represents H, C₆₋₁₀ aryl, C₁₋₁₀alkyl which latter group is optionally substituted by one or more halogroups, C₁₋₃ alkylphenyl, —C(R^(9a))(R^(9b))R¹⁰, —C(O)R^(11a),—C(O)OR¹², —C(O)N(R¹³)R¹⁴ or —(CH₂)_(n)(O)_(m)R¹⁵; R⁸ represents—C(R^(9a))(R^(9b))R¹⁰, —C(O)R^(11b) or —C(O)OR¹²; R^(9a) and R^(9b)independently represent, at each occurrence, H or C₁₋₆ alkyl; R¹⁰represents, at each occurrence, —OC(O)R^(16a), —OC(O)OR¹⁷,—N(R^(18a))C(O)OR¹⁷ or —OC(O)N(R^(18b))R¹⁷; R^(11a) and R^(11b)independently represent, at each occurrence, C₆₋₁₀ aryl, C₁₋₃alkylphenyl which latter two groups are optionally substituted by one ormore substituents selected from C₁₋₆ alkyl and halo,—[C(R^(19a))(R^(19b))]_(p)OC(O)R^(16b), or R^(11a) represents C₁₋₁₇alkyl optionally substituted by C₁₋₆ alkoxy, C₁₋₆ acyloxy, amino or haloor R^(11b) represents C₁₋₆ alkyl; R¹² represents, at each occurrence,C₁₋₁₇ alkyl optionally substituted by one or more substituents selectedfrom C₁₋₆ alkoxy, C₁₋₆ acyloxy, —Si(R^(20a))(R^(20b))(R^(20c)) and halo,C₆₋₁₀ aryl, C₁₋₃ alkylphenyl which latter two groups are optionallysubstituted by C₁₋₆ alkyl, C₁₋₆ alkoxy, cyano or halo,—[C(R^(19a))(R^(19b))]_(q)OC(O)R^(16b) or —CH₂R²¹; R¹³ represents H orC₁₋₇ alkyl, or together with R¹⁴ represents C₄₋₅ alkylene; R¹⁴represents C₆₋₁₀ aryl or C₁₋₁₀ alkyl which latter group is optionallysubstituted by one or more substituents selected from OH, halo, CO₂H,C₁₋₆ alkoxy, C₁₋₆ acyloxy and C₆₋₁₀ aryl, or together with R¹³represents C₄₋₅ alkylene; R¹⁵ represents C₁₋₇ alkyl optionallysubstituted by one or more —OC(O)C(H)(R²²)N(G)(G^(a)) groups; R^(16a),R^(16b) and R¹⁷ independently represent, at each occurrence, C₆₋₁₀ arylor C₁₋₁₇ alkyl which latter group is optionally substituted by one ormore substituents selected from —OH, halo, —CO₂H, C₁₋₆ alkoxy, C₁₋₆acyloxy and C₆₋₁₀ aryl, or R^(16b) represents C₁₋₆ alkoxy optionallysubstituted by one or more substituents selected from C₁₋₆ alkyl andhalo; R^(18a) and R^(18b) independently represent H or C₁₋₄ alkyl;R^(19a) and R^(19b) independently represent, at each occurrence, H orC₁₋₆ alkyl; R^(20a) to R^(20c) independently represent, at eachoccurrence, C₁₋₆ alkyl or phenyl; R²¹ represents the structural fragmentIIf

R²² represents C₃₋₄ alkyl; G and G^(a) independently represent H, anamino protective group, or G and G^(a) together represent an aminoprotective group; m represents 0 or 1; n represents 1, 2 or 3; prepresents 3 or 4; q represents 2 or 3; or a pharmaceutically acceptablesalt thereof, provided that: (a) D¹ and D² do not both represent H; and(b) when one of D¹ and D² represents —OR⁷, then the other represents H.9. A compound as defined in claim 1, wherein the fragment

is in the S-configuration.
 10. A compound as claimed in claim 1, whereinthe fragment

is in the R-configuration.
 11. A process for the preparation of acompound as claimed in claim 8, which comprises: (1) for compounds offormula IA in which one of D¹ and D² represents —OR⁷ in which R⁷represents H, C₆₋₁₀ aryl, C₁₋₁₀ alkyl which latter group is optionallysubstituted by one or more halo groups, or C₁₋₃ alkylphenyl, reaction ofa compound of formula XIV,

wherein Y, R¹ and R² are as defined in claim 8, with a compound offormula XV, H₂NOR^(a)  XV wherein R^(a) represents H, C₆₋₁₀ aryl, C₁₋₁₀alkyl which latter group is optionally substituted by one or more halogroups, or C₁₋₃ alkylphenyl, optionally by pre-treating the compound offormula XIV with gaseous HCl, in the presence of a lower alkyl alcoholto form a compound of formula XVI,

wherein R^(b) represents lower alkyl and Y, R¹ and R² are as defined inclaim 8; (2) for compounds of formula IA in which one of D¹ and D²represents —OR⁷ in which R⁷ represents H, C₆₋₁₀ aryl, C₁₋₁₀ alkyl whichlatter group is optionally substituted by one or more halo groups, orC₁₋₃ alkylphenyl, reaction of a corresponding compound of formula IA, inwhich one of D¹ and D² represents —C(O)OR¹² and the other represents H,with a compound of formula XV, as defined above, followed by removal ofthe —C(O)OR¹² group; (3) for compounds of formula IA in which D¹ or D²represents R⁸, reaction of a corresponding compound of formula I, or acorresponding compound of formula IA in which D¹ or D² as appropriaterepresents H, with a compound of formula XVII, L³—R⁸  XVII wherein L³represents a leaving group and R⁸ is as defined in claim 8; (4) forcompounds of formula IA in which one of D¹ and D² represents —OR⁷wherein R⁷ does not represent H, reaction of a corresponding compound offormula IA in which one of D¹ and D² represents —OH with a compound offormula XVIII,  L³—R^(7a)  XVIII wherein R^(7a) represents R⁷ as definedin claim 8, except that it does not represent H, and L³ is as definedabove; (5) for compounds of formula IA in which one of D¹ and D²represents H and the other represents —C(R^(9a))(R^(9b))R¹⁰, wherein R¹⁰represents —OC(O)R^(16a), —OC(O)OR¹⁷ or —OC(O)N(R^(18b))R¹⁷, reaction ofa corresponding compound of formula XIX,

wherein Y, R¹, R², R^(9a) and R^(9b) are as defined in claim 8, with acompound of formula XX, L³—C(O)R^(c)  XX wherein R^(c) representsR^(16a), —OR¹⁷ or —N(R^(8b))R¹⁷, and R^(16a), R¹⁷ and R^(18b) are asdefined in claim 8 and L³ is as defined above; (6) for compounds offormula IA in which D¹, D² and R³, together with the amidine group towhich they are attached, represent a group IIa as defined in claim 8,reaction of a corresponding compound of formula IA in which D¹represents OH with a compound of formula XXII, HalC(O)CH(R⁴)Hal  XXIIwherein Hal represents halo and R⁴ is as defined in claim 8, followed bycyclisation of the resultant intermediate; (7) for compounds of formulaIA in which D¹, D² and R³, together with the amidine group to which theyare attached, represent a group IIb as defined in claim 8, reaction of acorresponding compound of formula XXIII,

wherein Y, R¹ and R² are as defined in claim 8, with a compound offormula XXIV, H₂NCH(R⁴)C(O)OR^(b)  XXIV wherein R^(b) is as definedabove and R⁴ is as defined in claim 8; (8) for compounds of formula IAin which D¹, D² and R³, together with the amidine group to which theyare attached, represent a group IIc as defined in claim 8, reaction of acorresponding compound of formula IA in which D¹ represents OH with acompound of formula XXV, R⁴CHO  XXV wherein R⁴ is as defined in claim 8;(9) for compounds of formula IA in which D¹, D² and R³, together withthe amidine group to which they are attached, represent a group IId asdefined in claim 8, cyclisation of a compound of formula XXVI,

wherein Hal is as defined above and Y, R¹, R², R⁴, R⁵ and R⁶ are asdefined in claim 8; (10) for compounds of formula IA in which D¹, D² andR³, together with the amidine group to which they are attached,represent a group IIe as defined in claim 8, reaction of a correspondingcompound of formula XXIX,

wherein Y, R¹, R² and R⁶ are as defined in claim 8 with formaldehyde;(11) deprotection of a protected compound of formula IA as defined inclaim 8; or (12) introduction or interconversion of a substituent on anaromatic and/or non-aromatic, carbocyclic and heterocyclic ring(s) incompounds of formula IA as defined in claim
 8. 12. A pharmaceuticalcomposition comprising a compound as claimed in claim 1, or apharmaceutically acceptable salt or prodrug thereof, in admixture with apharmaceutically acceptable adjuvant, diluent or carrier.
 13. A methodof treatment of a condition where inhibition of thrombin is requiredwhich method comprises administration of a therapeutically effectiveamount of a compound as claimed in claim 1, or a pharmaceuticallyacceptable salt or prodrug thereof, to a person suffering from, orsusceptible to, such a condition.
 14. A method as claimed in claim 13,wherein the condition is thrombosis.
 15. A method as claimed in claim13, wherein the condition is hypercoagulability in blood and/or tissues.